Generation of acyl alcohols

ABSTRACT

Methods, compositions, and cells for generating acyl alcohols. Compositions comprising acyl alcohols. Methods of cleaving acyl amino acids and/or acyl alcohols to generate free fatty acids, free amino acids, and/or free alcohols.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to and benefit of U.S.provisional patent application No. 62/191,571, filed Jul. 13, 2015, theentire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 24, 2022, isnamed 2003320-0160_SL.txt and is 28,839 bytes in size.

BACKGROUND

Surfactants are currently manufactured from nonrenewable feedstocks(such as petroleum) or are manufactured from seed oils (such as palmoil), which contributes to rain forest destruction, as land is clearedto support palm plantation expansion. Use of raw materials to makesurfactants also increases greenhouse gas pollution. In addition,current surfactant manufacturing methods require enormous quantities ofheat, depend on hazardous processing steps (such as chlorination) andproduce toxic byproducts and carcinogens.

For example, cocamide monoethanolamine (MEA) and cocamide diethanolamine(DEA) are widely used commercially as nonionic surfactants. Thesesurfactants are typically produced by reacting fatty acid derived fromcoconut oil or palm oil with ethanolamine or diethanolamine.Ethanolamine and diethanolamine are produced on an industrial scale byreacting ethylene oxide with ammonia. The ethoxylation processes used tomanufacture cocamide MEA and cocamide DEA result in the generation ofthe carcinogen 1,4-dioxane as a contaminant.

SUMMARY

In one aspect, the present disclosure encompasses the recognition thatthere is a need for “greener” methods of producing surfactants(including non-ionic surfactants), e.g., methods that do not lead to theformation of toxic substances. In one aspect, the present disclosureprovides methods of generating acyl alcohols.

In certain embodiments, provided are methods comprising steps of:providing an acyl amino acid and treating the acyl amino acid with areductase polypeptide so that an acyl alcohol is released. In someembodiments, both the providing and treating stems are performed in acell expressing a reductase polypeptide that acts on the acyl aminoacid. In some embodiments, the acyl amino acid is generated in the cell.

In some embodiments, the cell expresses an engineered polypeptidecomprising a fatty acid linkage domain, a peptide synthetase domain, anda first reductase polypeptide. In some embodiments, the fatty acidlinkage domain and the peptide synthetase domain are covalently linked.In some embodiments, the reductase polypeptide is covalently linked tothe fatty acid linkage domain and the peptide synthetase domain. In someembodiments, the fatty acid linkage domain is a beta-hydroxy fatty acidlinkage domain, e.g., a beta-hydroxy myristic acid linkage domain.

In some embodiments, the peptide synthetase domain comprises anadenylation domain and a thiolation domain, which adenylation domain andthiolation domain are covalently linked. In some embodiments, theadenylation domain is specific for glycine. In some embodiments, theadenylation domain is at least 70% identical at the amino acid level tothe terminal adenylation domain of the gramicidin peptide synthetasefrom Bacillus brevis.

In some embodiments, the cell expresses a second reductase polypeptidedistinct from the first reductase polypeptide. In some embodiments, thesecond reductase polypeptide is at least 70% identical at the amino acidlevel to the polypeptide produced from Bacillus brevis LgrE gene.

In some embodiments, the acyl alcohol is acyl ethanolamine, e.g.,β-hydroxy myristoyl ethanolamine.

In some embodiments, the adenylation domain is specific for alanine. Insome embodiments, the adenylation domain is at least 70% identical atthe amino acid level to the reductase domain of the terminalmycobacterial glycopeptidedolipid peptide synthetase domain.

In some embodiments, the acyl alcohol is acyl alaninol.

In some embodiments, the adenylation domain is specific for serine.

In some embodiments, the acyl alcohol is acyl serinol.

In some embodiments, the cells are grown in a liquid media. In someembodiments, the acyl alcohol is secreted into the liquid media.

In some embodiments, the cell is a microbial cell, e.g., a Bacilluscell. In some embodiments, the Bacillus cell is a Bacillus subtiliscell.

In one aspect, the present disclosure compasses the recognition that,although acyl amino acids and acyl amino alcohols have commercial value,it may be desirable to produce derivative products from acyl amino acidsor acyl amino alcohols, which may have commercial value in a widermarket. In one aspect, the present disclosure provides methods ofcleaving an acyl amino acid and/or acyl alcohol.

In certain embodiments, provided methods comprise steps of providing anacyl amino acid and/or an acyl alcohol and treating the acyl amino acidor acyl alcohol so that i) free fatty acid and ii) free amino acidand/or free alcohol is released.

In some embodiments, the treating step comprises incubating the acylamino acid or acyl alcohol in acid. In some embodiments, the acyl aminoacid or acyl alcohol is incubated in acid with heat.

In some embodiments, the treating step comprises incubating the acylamino acid with an enzyme.

In some embodiments, methods further comprise a step of separating thereleased free fatty acid from the released free amino acid or freealcohol.

In some embodiments, methods further comprise a step of treating thefree fatty acid so as to reduce the oxygen content of the free fattyacid.

In some embodiments, the free fatty acid is myristic acid.

In one aspect, provided are compositions comprising an acyl alcoholcomprising a fatty acid covalently and directly linked to an alcohol. Insome embodiments, the fatty is linked to the alcohol via an amide bond.In some embodiments, the a alcohol is an amino alcohol.

In one aspect, provided are compositions comprising an acyl alcohol andone or more components of an engineered microbial cell. In someembodiments, the one or more components comprise an intact microbialcell.

In one aspect, provided are engineered microbial cells comprising one ormore polypeptides collectively comprising: a fatty acid linkage domain,a peptide synthetase domain, and one or more reductase polypeptides,which fatty acid linkage domain is covalently linked to the peptidesynthetase domain, which fatty acid linkage domain and peptidesynthetase domain collectively can produce an acyl amino acid, and whichone or more reductase polypeptides are collectively capable of reducingthe acyl amino acid to an acyl alcohol. In some embodiments, theengineered microbial cell lacks a thioesterase domain. In someembodiments, the microbial cell is a bacterial cell. In someembodiments, the bacterial cell is a Bacillus cell. In some embodiments,the Bacillus cell is a Bacillus subtilis cell.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the structures of cocamide diethanolamine (DEA), cocamidemonoethanolamine (MEA), and beta-hydroxyl myristoyl monoethanolamine(MEA).

FIG. 2 depicts a schematic showing domains involved in the synthesis ofsurfactin by the surfactin synthetase from Bacillus subtilis.

FIG. 3 depicts a schematic showing a mini-synthetase that links fattyacid to glutamic acid.

FIG. 4 shows the amino acid sequence of SEQ ID NO: 3, corresponding to aportion of module 16 of the Bacillus brevis linear gramicidin synthetasecomplex. This sequence was obtained as described in Example 1 andincludes the condensation, adenylation, thiolation and reductase domainsof module 16. Underlining marks the beginning of the reductase domainaccording to Manavalan et al. (“Molecular modeling of the reductasedomain to elucidate the reaction mechanism of reduction of peptidylthioester into its corresponding alcohol in non-ribosomal peptidesynthetases.” BMC Structural Biology. 2010, 10:1. DOI:10.1186/1472-6807-10-1, (the entire contents of which are hereinincorporated by reference)), bold marks the NADPH-binding domain, andsquare boxes marks catalytic residues. A conserved adenylation domainsequence is bolded and underlined.

FIGS. 5A and 5B show the amino acid sequence (SEQ ID NO: 4) andnucleotide (coding) sequence (SEQ ID NO: 5), respectively, of theadenylation domain within SEQ ID NO: 3. In FIG. 5A, a conservedadenylation domain sequence is bolded and underlined.

FIGS. 6A and 6B show the amino acid sequence (SEQ ID NO: 6) andnucleotide sequence (SEQ ID NO: 7), respectively, of the lgrE gene fromBacillus brevis.

FIG. 7, comprising panels A and B, depict results from experimentsvalidating a quantitative LCMS method to analyze a commercialpreparation of cocoyl glycinate. FIG. 7, panel A depicts LC/MS results,in which peaks corresponding to C12, C14, and C16 are visible. FIG. 7,panel B (inset) depicts the LC/MS signal plotted as a function of theamount of surfactant.

FIG. 8, comprising panels A and B, depicts results from the LC/MSexperiment described in Example 3 analyzing derivative products from areaction in which FA-Glu (an acyl amino acid) was treated with acid.FIG. 8, panel A, shows the results when FA-Glu was injected into theanalyzer without any treatment. FIG. 8, panel B shows that a strongglutamate peak was detected after FA-Glu had been incubated with acid.

FIG. 9 (comprising panels A, B, C), FIG. 10 (comprising panels A, B, C),FIG. 11 (comprising panels A, B, C), FIG. 12 (comprising panels A, B,C), FIG. 13 (comprising panels A, B, C), and FIG. 14 (comprising panelsA, B. and C) depict results from an LC/MS experiment described inExample 4 analyzing derivative products from FA-Glu treated with anacylase. FIGS. 9, 10, 11, 12, and 13 show chromatograms for thedetection of beta-hydroxy fatty acid compounds and FA-Glu for m/zrations of between 200 to 900, between 190 to 300, between 440 to 558,between 320 to 430, and between 682 to 848, respectively. FIG. 14 showschromatograms for the detection of glutamate. Panel A in each figuredepicts LC/MS data for FA-Glu incubated with porcine kidney acylase I,panel B in each figure depicts LC/MS data for porcine kidney acylase Iincubated without FA-Glu, and panel C in each figure depicts LC/MS datafor FA-Glu incubated without porcine kidney acylase I.

DEFINITIONS

In this application, unless otherwise clear from context, (i) the term“a” may be understood to mean “at least one”; (ii) the term “or” may beunderstood to mean “and/or”; (iii) the terms “comprising” and“including” may be understood to encompass itemized components or stepswhether presented by themselves or together with one or more additionalcomponents or steps; and (iv) the terms “about” and “approximately” maybe understood to permit standard variation as would be understood bythose of ordinary skill in the art; and (v) where ranges are provided,endpoints are included.

As used herein, the term “acyl amino acid” refers to an amino acid thatis covalently linked to a fatty acid. In certain embodiments, acyl aminoacids produced by compositions and methods as described, e.g., in U.S.Pat. No. 7,981,685, the entire contents of which are incorporated byreference herein. In certain embodiments, acyl amino acids are producedby employing engineered polypeptides comprising a peptide synthetasedomain covalently linked to a fatty acid linkage domain. In certainembodiments, the fatty acid linkage domain is a beta-hydroxy fatty acidlinkage domain. Typically, the identity of the amino acid moiety of theacyl amino acid is determined by the amino acid specificity of thepeptide synthetase domain, and in particular by the adenylation domainwithin the peptide synthetase domain. For example, the peptidesynthetase domain may specify any one of the naturally occurring aminoacids known by those skilled the art to be used in ribosome-mediatedpolypeptide synthesis. Alternatively, the peptide synthetase domain mayspecify a non-naturally occurring amino acid, e.g. a modified aminoacid. Similarly, the identity of the fatty acid moiety of the acyl aminoacid is determined by the fatty acid specificity of the fatty acidlinkage domain, such as for example a fatty acid linkage domain that isspecific for a beta-hydroxy fatty acid. For example, the beta-hydroxyfatty acid may be any of a variety of naturally occurring ornon-naturally occurring beta-hydroxy fatty acids.

As used herein, the term “acyl alcohol” refers to a compound in which analcohol is covalently and directly linked to a fatty acid. As usedherein, the term “acyl amino alcohol” refers to a type of acyl alcoholin which the alcohol that is covalently and directly linked to the fattyacid is an amino alcohol. In accordance with certain methods disclosedherein, an acyl alcohol is generated by treating an acyl amino acid withone or more reductase polypeptides such that an acyl alcohol isreleased. The acyl amino acid from which the acyl alcohol is generatedand released can contain a naturally occurring amino acid or anon-naturally occurring amino acid. Possible fatty acid moieties of anacyl alcohol or an acyl amino alcohol include any fatty acid that can bespecified by a fatty acid linkage domain (whether naturally occurring orengineered) of a peptide synthetase complex. In some embodiments, thefatty acid moiety is a beta-hydroxy fatty acid moiety, e.g.,beta-myristic acid.

As used herein, the term “beta-hydroxy fatty acid” refers to a fattyacid chain comprising a hydroxy group at the beta position of the fattyacid chain. As is understood by those skilled in the art, the betaposition corresponds to the third carbon of the fatty acid chain, thefirst carbon being the carbon of the carboxylate group. Thus, when usedin reference to an acyl amino acid of the present disclosure, where thecarboxylate moiety of the fatty acid has been covalently attached to thenitrogen of the amino acid, the beta position corresponds to the carbontwo carbons removed from the carbon having the ester group. Abeta-hydroxy fatty acid to be used in accordance with the presentdisclosure may contain any number of carbon atoms in the fatty acidchain. As non-limiting examples, a beta-hydroxy fatty acid may contain3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 3, 14, 15, 15, 16, 17, 18, 19, 20 ormore carbon atoms. Beta-hydroxy fatty acids to be used in accordancewith the present disclosure may contain linear carbon chains, in whicheach carbon of the chain, with the exception of the terminal carbon atomand the carbon attached to the nitrogen of the amino acid, is directlycovalently linked to two other carbon atoms. Additionally oralternatively, beta-hydroxy fatty acids to be used in accordance withthe present disclosure may contain branched carbon chains, in which atleast one carbon of the chain is directly covalently linked to three ormore other carbon atoms. Beta-hydroxy fatty acids to be used inaccordance with the present disclosure may contain one or more doublebonds between adjacent carbon atoms. Alternatively, beta-hydroxy fattyacids to be used in accordance with the present disclosure may containonly single-bonds between adjacent carbon atoms. A non-limitingexemplary beta-hydroxy fatty acid that may be used in accordance withthe present disclosure is beta-hydroxy myristic acid, which contains 13to 15 carbons in the fatty acid chain. Those of ordinary skill in theart will be aware of various beta-hydroxy fatty acids that can be usedin accordance with the present disclosure. Different beta-hydroxy fattyacid linkage domains that exhibit specificity for other beta-hydroxyfatty acids (e.g., naturally or non-naturally occurring beta-hydroxyfatty acids) may be used in accordance with the present disclosure togenerate any acyl amino acid of the practitioner's choosing.

As used herein, the term “covalently linked” refers to its ordinarymeaning in the art, and unless otherwise specified, encompasses bothdirect and indirect linkages.

As used herein, the terms “domain” and “polypeptide domain” generallyrefer to polypeptide moieties that naturally occur in longerpolypeptides, or to engineered polypeptide moieties that are homologousto such naturally occurring polypeptide moieties, which polypeptidemoieties have a characteristic structure (e.g., primary structure suchas the amino acid sequence of the domain, although characteristicstructure of a given domain also encompasses secondary, tertiary,quaternary, etc. structures) and exhibit one or more distinct functions.As will be understood by those skilled in the art, in many cases,polypeptides are modular and are comprised of one or more polypeptidedomains, each domain exhibiting one or more distinct functions thatcontribute to the overall function of the polypeptide. The structure andfunction of many such domains are known to those skilled in the art. Forexample, Fields and Song (Nature, 340(6230): 245-6, 1989) showed thattranscription factors are comprised of at least two polypeptide domains:a DNA binding domain and a transcriptional activation domain, each ofwhich contributes to the overall function of the transcription factor toinitiate or enhance transcription of a particular gene that is undercontrol of a particular promoter sequence. A polypeptide domain, as theterm is used herein, also refers an engineered polypeptide that ishomologous to a naturally occurring polypeptide domain. “Homologous”, asthe term is used herein, refers to the characteristic of being similarat the nucleotide or amino acid level to a reference nucleotide orpolypeptide. For example, a polypeptide domain that has been altered atone or more positions such that the amino acids of the referencepolypeptide have been substituted with amino acids exhibiting similarbiochemical characteristics (e.g., hydrophobicity, charge, bulkiness)will generally be homologous to the reference polypeptide. Percentidentity and similarity at the nucleotide or amino acid level are oftenuseful measures of whether a given nucleotide or polypeptide ishomologous to a reference nucleotide or amino acid. Those skilled in theart will understand the concept of homology and will be able todetermine whether a given nucleotide or amino acid sequence ishomologous to a reference nucleotide or amino acid sequence.

As used herein, the term “engineered” refers to a non-naturallyoccurring moiety that has been created by the hand of man. For example,in reference to a polypeptide, an “engineered polypeptide” refers to apolypeptide that has been designed and/or manipulated to comprise apolypeptide that does not exist in nature. In various embodiments, anengineered polypeptide comprises two or more covalently linkedpolypeptide domains. Typically such domains will be linked via peptidebonds, although the present disclosure is not limited to engineeredpolypeptides comprising polypeptide domains linked via peptide bonds,and encompasses other covalent linkages known to those skilled in theart. One or more covalently linked polypeptide domains of engineeredpolypeptides may be naturally occurring. Thus, in certain embodiments,engineered polypeptides of the present disclosure comprise two or morecovalently linked domains, at least one of which is naturally occurring.In certain embodiments, two or more naturally occurring polypeptidedomains are covalently linked to generate an engineered polypeptide. Forexample, naturally occurring polypeptide domains from two or moredifferent polypeptides may be covalently linked to generate anengineered polypeptide. In certain embodiments, naturally occurringpolypeptide domains of an engineered polypeptide are covalently linkedin nature, but are covalently linked in the engineered polypeptide in away that is different from the way the domains are linked nature. Forexample, two polypeptide domains that naturally occur in the samepolypeptide but which are separated by one or more intervening aminoacid residues may be directly covalently linked (e.g., by removing theintervening amino acid residues) to generate an engineered polypeptideof the present disclosure. Additionally or alternatively, twopolypeptide domains that naturally occur in the same polypeptide whichare directly covalently linked together (e.g., not separated by one ormore intervening amino acid residues) may be indirectly covalentlylinked (e.g., by inserting one or more intervening amino acid residues)to generate an engineered polypeptide of the present disclosure. Incertain embodiments, one or more covalently linked polypeptide domainsof an engineered polypeptide may not exist naturally. For example, suchpolypeptide domains may be engineered themselves.

As used herein, the term “fatty acid linkage domain” refers to apolypeptide domain that covalently links a fatty acid to an amino acidto form an acyl amino acid. In certain embodiments, a fatty acid linkagedomain is covalently linked to one or more subdomains of a peptidesynthetase domain to generate an engineered polypeptide useful in thesynthesis of an acyl amino acid. A variety of fatty acids are known tothose of ordinary skill in the art, as are a variety of fatty acidlinkage domains, such as for example, fatty acid linkage domains presentin various peptide synthetase complexes that produce lipopeptides. Incertain embodiments, a fatty acid linkage domain of the presentdisclosure comprises a beta-hydroxy fatty acid linkage domain.

As used herein, the term “identity,” refers to the overall relatednessbetween polymeric molecules, e.g., between nucleic acid molecules (e.g.,DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. In some embodiments, polymeric molecules are considered to be“substantially identical” to one another if their sequences are at least25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% identical. Calculation of the percent identity of twonucleic acid or polypeptide sequences, for example, can be performed byaligning the two sequences for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second sequences foroptimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or substantially 100% of the length of a reference sequence. Thenucleotides or amino acid residues at corresponding positions are thencompared. When a position in the first sequence is occupied by the sameresidue (e.g., nucleotide or amino acid) as the corresponding positionin the second sequence, then the molecules are identical at thatposition. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences, takinginto account the number of gaps, and the length of each gap, which needsto be introduced for optimal alignment of the two sequences. Thecomparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm.

As used herein, the term “isolated” refers to a substance and/or entitythat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in natureand/or in an experimental setting), and/or (2) designed, produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. In some embodiments, as willbe understood by those skilled in the art, a substance may still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. To give but one example, in someembodiments, a biological polymer such as a polypeptide orpolynucleotide that occurs in nature is considered to be “isolated”when, a) by virtue of its origin or source of derivation is notassociated with some or all of the components that accompany it in itsnative state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; c) is expressed by or is otherwise in associationwith components from a cell or other expression system that is not ofthe species that produces it in nature. Thus, for instance, in someembodiments, a polypeptide that is chemically synthesized or issynthesized in a cellular system different from that which produces itin nature is considered to be an “isolated” polypeptide. Alternativelyor additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques may be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

As used herein, the term “naturally occurring”, as used herein whenreferring to an amino acid, refers to one of the standard group oftwenty amino acids that are the building blocks of polypeptides of mostorganisms, including alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. In certain embodiments, the term“naturally occurring” also refers to amino acids that are used lessfrequently and are typically not included in this standard group oftwenty but are nevertheless still used by one or more organisms andincorporated into certain polypeptides. For example, the codons UAG andUGA normally encode stop codons in most organisms. However, in someorganisms the codons UAG and UGA encode the amino acids selenocysteineand pyrrolysine. Thus, in certain embodiments, selenocysteine andpyrrolysine are naturally occurring amino acids. The term “naturallyoccurring”, as used herein when referring to a polypeptide orpolypeptide domain, refers to a polypeptide or polypeptide domain thatoccurs in one or more organisms. In certain embodiments, engineeredpolypeptides of the present disclosure comprise one or more naturallyoccurring polypeptide domains that naturally exist in differentpolypeptides. In certain embodiments, engineered polypeptides of thepresent disclosure comprise two or more naturally occurring polypeptidedomains that are covalently linked (directly or indirectly) in thepolypeptide in which they occur, but are linked in the engineeredpolypeptide in a non-natural manner. As a non-limiting example, twonaturally occurring polypeptide domains that are directly covalentlylinked may be separated in the engineered polypeptide by one or moreintervening amino acid residues. Additionally or alternatively, twonaturally occurring polypeptide domains that are indirectly covalentlylinked may be directly covalently linked in the engineered polypeptide,e.g. by removing one or more intervening amino acid residues. Suchengineered polypeptides are not naturally occurring, as the term is usedherein.

As used herein, the term “operably linked” refers to a relationshipbetween two nucleic acid sequences wherein the expression of one of thenucleic acid sequences is controlled by, regulated by or modulated bythe other nucleic acid sequence. In some embodiments, a nucleic acidsequence that is operably linked to a second nucleic acid sequence iscovalently linked, either directly or indirectly, to such secondsequence, although any effective three-dimensional association isacceptable. A single nucleic acid sequence can be operably linked tomultiple other sequences. For example, a single promoter can directtranscription of multiple RNA species.

As used herein, the term “peptide synthetase domain” refers to apolypeptide domain that typically comprises three domains: anadenylation (A) domain, responsible for selectively recognizing andactivating a specific amino acid, a thiolation (T) domain, which tethersthe activated amino acid to a cofactor via thioester linkage, andcondensation (C) domain, which links amino acids joined to successiveunits of the peptide synthetase by the formation of amide bonds. Apeptide synthetase domain typically recognizes and activates a single,specific amino acid, and in the situation where the peptide synthetasedomain is not the first domain in the pathway, links the specific aminoacid to a growing peptide chain. In certain embodiments, a peptidesynthetase domain is covalently linked to a fatty acid linkage domainsuch as a beta-hydroxy fatty acid linkage domain, which construct may beadvantageously used to generate an acyl amino acid. In certainembodiments, the peptide synthetase domain comprises fewer than thethree typical domains. For example, a peptide synthetase domain inaccordance with certain methods and compositions of the disclosure mayinclude the adenylation and thiolation domains covalently linked to eachother, but may lack a condensation domain. In certain engineeredpolypeptides of the disclosure, for example, the condensation domain ofthe peptide synthetase is not present, and/or a different domain (e.g.,a fatty acid linkage domain) is covalently linked to the adenylation andthiolation domains. In certain embodiments, a peptide synthetase domainis also covalently linked to a reductase domain that acts on the acylamino acid so produced, as described herein. A variety of peptidesynthetase domains are known to those skilled in the art, e.g. such asthose present in a variety of nonribosomal peptide synthetase complexes.Those skilled in the art will be aware of methods to determine whether agive polypeptide domain is a peptide synthetase domain. Differentpeptide synthetase domains often exhibit specificity for one or moreamino acids. As one non-limiting example, the last peptide synthetasedomain from the gramicidin peptide synthetase is specific for glycine.Thus, the last peptide synthetase domain from gramicidin peptidesynthetase can be used in methods known in the art (e.g., as describedin U.S. Pat. No. 7,981,685) to construct an engineered polypeptideuseful in the generation of an acyl amino acid that comprises a glycinemoiety, which, in accordance with certain methods of the presentdisclosure, can be treated with one or more reductase polypeptides sothat acyl ethanolamine (FA-ethanolamine), a non-ionic surfactant isreleased. Different peptide synthetase domains that exhibit specificityfor other amino acids (e.g., naturally or non-naturally occurring aminoacids) may be used in accordance with methods known in the art togenerate any acyl amino acid, which acyl amino acid can then be treatedin accordance with methods of the disclosure to generate any of avariety of acyl alcohols of the practitioner's choosing.

As used herein, the term “polypeptide” refers to a series of amino acidsjoined together in peptide linkages, such as polypeptides synthesized byribosomal machinery in naturally occurring organisms. The term“polypeptide” also refers to a series of amino acids joined together bynon-ribosomal machinery, such as by way of non-limiting example,polypeptides synthesized by various peptide synthetases, including bothnaturally occurring and engineered peptide synthetases. Suchnon-ribosomally produced polypeptides exhibit a greater diversity incovalent linkages than polypeptides synthesized by ribosomes (althoughthose skilled in the art will understand that the amino acids ofribosomally-produced polypeptides may also be linked by covalent bondsthat are not peptide bonds, such as the linkage of cysteines viadi-sulfide bonds). For example, surfactin is a lipopeptide synthesizedby the surfactin synthetase complex (a schematic of which is depicted inFIG. 2). Surfactin comprises seven amino acids, which are initiallyjoined by peptide bonds, as well as a beta-hydroxy fatty acid covalentlylinked to the first amino acid, glutamate. However, upon addition thefinal amino acid (leucine), the polypeptide is released and thethioesterase domain of the SRFC protein catalyzes the release of theproduct via a nucleophilic attack of the beta-hydroxy of the fatty acidon the carbonyl of the C-terminal Leu of the peptide, cyclizing themolecule via formation of an ester, resulting in the C-terminus carboxylgroup of leucine attached via a lactone bond to the b-hydroxyl group ofthe fatty acid. Polypeptides can be two or more amino acids in length,although most polypeptides produced by ribosomes and peptide synthetasesare longer than two amino acids. For example, polypeptides may be 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 or moreamino acids in length.

As used herein, the term “reductase polypeptide” refers to anypolypeptide that 1) catalyzes reduction of an amino acid, or reductionproduct of an amino acid (e.g., an amino aldehyde) attached by a peptidesynthetase complex and/or 2) releases a product of the peptidesynthetase complex with the terminal residue as a reduction product ofan amino acid (e.g., an amino aldehyde or an alcohol such as an aminoalcohol) from the peptide synthetase complex. In certain embodiments, areductase polypeptide is a polypeptide domain that is covalently linkedto one or more other domains, e.g., it is a “reductase domain” within alarger polypeptide comprising more than one polypeptide domain. In someembodiments, the reductase polypeptide and the one or more other domainsto which it is covalently linked form an engineered polypeptide. Forexample, in some embodiments, the reductase polypeptide is a reductasedomain that is covalently linked to one or more subdomains of a peptidesynthetase domain and a fatty acid linkage domain such as a beta-hydroxyfatty acid linkage domain to generate an engineered polypeptide usefulin the synthesis of an acyl alcohol, e.g., an acyl amino alcohol.Accordingly, in some embodiments, a reductase polypeptide catalyzesreduction of an acyl amino acid, or a reduction product thereof, and/orrelease a reduction product of an acyl amino acid (e.g., an acyl aminoaldehyde or an acyl alcohol, e.g., acyl amino alcohol.) A variety ofreductase domains are found in nonribosomal peptide synthetase complexesfrom a variety of species. A non-limiting example of a reductase domainthat may be used in accordance with the present disclosure includes thereductase domain from linear gramicidin (ATCC8185). However, anyreductase polypeptide that at least partially reduces an acyl amino acidproduced by a peptide synthetase complex and/or releases a reduced acylamino acid from the peptide synthetase complex may be used in accordancewith the present disclosure. In some embodiments, reductase polypeptidesand reductase domains are characterized by the presence of the consensussequence:[LIVSPADNK]-x(9)-{P}-x(2)-Y-[PSTAGNCV]-[STAGNQCIVM]-[STAGC]-K-{PC}-[SAGFYR]-[LIVMSTAGD]-x-{K}-[LIVMFYW]-{D}-x-{YR}-[LIVMFYWGAPTHQ]-[GSACQRHM](SEQ ID NO: 1), where square brackets (“[ ]”) indicate amino acids thatare typically present at that position, squiggly brackets (“{ }”)indicate amino acids that amino acids that are typically not present atthat position, and “x” denotes any amino acid or a gap. X(9) for exampledenotes any amino acids or gaps for nine consecutive positions. Thoseskilled in the art will be aware of methods to determine whether a givepolypeptide domain is a reductase domain. Reductase polypeptidescompatible for use in presently disclosed methods include reductasepolypeptides with substantially the same sequence as a reductasepolypeptide found in nature as well as reductase polypeptides whosesequences are not found in nature. For example, some reductasepolypeptides suitable for use with certain of the presently disclosedmethods are engineered to have one or more mutations relative to asequence of a naturally occurring reductase polypeptides.

Detailed Description of Certain Embodiments

The present disclosure provides, in various aspects, methods ofgenerating acyl alcohols, methods of generating free fatty acids, freeamino acids, and/or free alcohols from acyl amino acids and/or acylalcohols, and related compositions and cells therefore.

Methods of Generating Acyl Alcohols

In one aspect, provided are methods comprising steps of providing anacyl amino acid and treating the acyl amino acid with a reductasepolypeptide so that an acyl alcohol is released.

In some embodiments, the steps of providing and treating are performedin a cell expressing one or more reductase polypeptides that act on theacyl amino acid. In some embodiments, the acyl amino acid is generatedin the cell.

In some embodiments, the cell expresses one or more engineeredpolypeptides, as described further herein.

In some embodiments, the acyl alcohol is a non-ionic surfactant.

Uses

In certain embodiments, compositions and methods of the presentdisclosure are useful in large-scale production of acyl alcohols. Incertain embodiments, acyl alcohols are produced in commercially viablequantities using compositions and methods of the present disclosure. Forexample, engineered polypeptides of the present disclosure may be usedto produce acyl alcohols to a level of at least 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1000 mg/L or higher. Biological production of acyl alcohols usingengineered polypeptides of the present disclosure achieves certainadvantages over other methods of producing acyl alcohols. For example,as compared to chemical production methods, production of acyl alcoholsusing compositions and methods of the present disclosure utilizes morereadily available and starting materials that are easier to store,reduces the necessity of using harsh and sometimes dangerous chemicalreagents in the manufacturing process, reduces the difficulty andefficiency of the synthesis itself by utilizing host cells asbioreactors, and reduces the fiscal and environmental cost of disposingof chemical by-products. For example, no oil is needed to produce acylalcohols according to methods of the present disclosure.

As a non-limiting example, using certain methods of the presentdisclosure, fatty acids can be linked to ethanolamine, producing an acylethanolamine that is a non-ionic surfactant and is similar to cocamidemonoethanolamine (MEA). In some embodiments, such an acyl alcohol isproduced by generating an acyl amino acid from engineered polypeptidecomprising fatty acid linkage domain that is covalently linked to anadenylation domain specific for glycine (e.g., an adenylation domainfrom or derived from a peptide synthetase domain specific for glycine)and a thiolation domain. The acyl glycine that is produced is then actedupon by one or more reductase polypeptides that may or may not becovalently liked to the fatty acid linkage, adenylation, and thiolationdomains, such that a fatty acid-ethanolamine is released from theengineered polypeptide.

As another non-limiting example, fatty acids can be linked to serinol(i.e., 2-amino-1-3-propanediol), producing chemical products similar tococamide diethanolamine (DEA). In some embodiments, such an acyl alcoholis produced by generating an acyl amino acid from engineered polypeptidecomprising fatty acid linkage domain that is covalently linked to anadenylation domain specific for serine (e.g., an adenylation domain fromor derived from a peptide synthetase domain specific for serine) and athiolation domain. The acyl serine that is produced is then acted uponby one or more reductase polypeptides that may or may not be covalentlyliked to the fatty acid linkage, adenylation, and thiolation domains,such that a fatty acid-serinol is released from the engineeredpolypeptide.

Methods of Cleaving Acyl Alcohols

In one aspect, provided are methods comprising steps of providing anacyl amino acid or acyl alcohol and treating the acyl amino acid or acylalcohol so that free fatty acid and free amino acid or free alcohol isreleased.

In certain embodiments, the treating step comprises incubating the acylamino acid or acyl alcohol in acid for a period of time. For example, insome embodiments, the acyl amino acid or acyl alcohol is incubated inacid for at least 30 minutes. In some embodiments, the acyl amino acidor acyl alcohol is incubated for at least 1, at least 2, at least 4, atleast 6, at least 8, at least 10, at least 12, at least 14, at least 16,at least 18, at least 20, at least 22, at least 24, at least 30, atleast 36, at least 42, or at least 48 hours. In some embodiments, theacyl amino acid or acyl alcohol is incubated in acid for at least orapproximately 12 hours. In some embodiments, the acyl amino acid or acylalcohol is incubated in acid for at least or approximately 24 hours. Insome embodiments, the acyl amino acid or acyl alcohol is incubated inacid for at least or approximately 36 hours. In some embodiments, theacyl amino acid or acyl alcohol is incubated in acid for at least orapproximately 48 hours.

In some embodiments, the acyl amino acid or acyl alcohol is incubated inacid in the presence of heat, i.e., such that the incubation temperatureis higher than that of room temperature (typically at or around 20° C.to at or around 26° C.), for at least a portion of the total acidincubation time. For example, in some embodiments, the acyl amino acidor acyl alcohol is incubated at a temperature of at least 30° C., atleast 35° C., at least 40° C., at least 45° C., at least 50° C., atleast 55° C., at least 60° C., at least 65° C., at least 70° C., atleast 75° C., at least 80° C., at least 85° C., at least 90° C., atleast 95° C., at least 100° C., at least 110° C., at least 120° C., atleast 130° C., at least 140° C., at least 150° C., at least 160° C., atleast 170° C., at least 180° C., at least 190° C., or at least 200° C.In some embodiments, the acyl amino acid or acyl alcohol is incubated ata temperature of at least or approximately 80° C. In some embodiments,the acyl amino acid or acyl alcohol is incubated at a temperature of atleast or approximately 100° C.

The concentration of the acid used may vary depending on the embodiment.In some embodiments, the concentration depends on factors such as any orany combination of the type of acid used, the period of incubation, andthe temperature.

In some embodiments, the acid is hydrochloric acid. In some embodiments,the hydrochloric acid is used at a concentration of at least 2 N, atleast 2.5 N, at least 3 N, at least 3.5 N, at least 4 N, at least 4.5 N,at least 5 N, at least 5.5 N, at least 6 N, at least 7 N, at least 8 N,at least 9 N, or at least 10 N. In some embodiments, the hydrochloricacid is used at a concentration of at least or approximately 6 N.

In certain embodiments, the treating step comprises incubating the acylamino acid or acyl alcohol with an enzyme for a period of time.Generally, such an enzyme is capable of cleaving the amide bond withinan acyl amino acid or acid alcohol. Examples of such enzymes include,but are not limited to, acylases, many types of which are commerciallyavailable. (See also, e.g., Otvos et al, “Investigation on the mechanismof acylase-I-catalyzed acyl amino acid hydrolysis,” Biochem Bipophys ResCommun 44(5):1056-1064, 1971, the entire contents of which areincorporated by reference herein.)

In certain embodiments, the treating step comprises incubating the acylamino acid or acyl alcohol at an elevated pressure (above that ofatmosphere) for a period of time. For example, the acyl amino acid oracyl alcohol can be incubating at a pressure of at least orapproximately 100 kPa (15 psi). In some embodiments, the acyl amino acidor acyl alcohol is incubated in a vacuum. In some embodiments, the acylamino acid or acyl alcohol can be incubated at both an elevated pressureand an elevated temperature (optionally also in a vacuum), e.g., as inan autoclave. (See, e.g., Badadani et al., Optimum conditions forautoclaving for hydrolysis of proteins and urinary peptides of prolyland hydroxyprolyl residues and HPLC analysis,” J Chromatogra B AnalytTechnol Biomed Life Sci 847(2):267-274, 2006, the entire contents ofwhich are incorporated by reference herein.)

Any suitable combination of the above-mentioned treating steps can alsobe used, either sequentially or concurrently.

In certain embodiments, only an acyl amino acid, or a mixture of acylamino acids having the same amino acid moiety and the same fatty acidmoiety (which can be of variable length), is provided. That is, in suchembodiments, no acyl alcohol is provided. In some embodiments, productsof cleavage would include free fatty acids and free amino acids.

In certain embodiments, only an acyl alcohol, or a mixture of acylalcohols having the same alcohol moiety and the same fatty acid moiety(which can be of variable length), is provided. That is, in suchembodiments, no acyl amino acid is provided. In some embodiments,products of cleavage would include free fatty acids and free alcohols.

In certain embodiments, a mixture of acyl amino acids and acyl alcoholsis provided. In some embodiments, products of cleavage would includefree fatty acids, free amino acids, and free alcohols.

In some embodiments, the free fatty acid is myristic acid.

In certain embodiments, provided methods further comprise a step ofseparating at least one cleavage product from other cleavage products.In some embodiments, provided methods further comprise a step ofseparating free fatty acids from other cleavage product(s). In someembodiments, provided methods further comprise a step of separating freeamino acids from other cleavage product(s). In some embodiments,provided methods further comprise a step of separating free alcoholsfrom other cleavage product(s). In some embodiments, a single step ofseparating allows the simultaneous recovery of more than one cleavageproduct. For example, phase separation can be used to separate freefatty acids from free amino acids and/or free alcohols, as fatty acid isnot highly soluble in water and forms a separate layer distinct from theaqueous layer.

In certain embodiments, provided methods further comprise a step oftreating the fatty acid so as to reduce the oxygen content of the freefatty acid, e.g., by hydrotreatment. (See, e.g., Marinangeli et al.,“Opportunities for biorenewables in oil refineries,” Submitted to U.S.Department of Energy 1-43, 2005, the entire contents of which areincorporated by reference herein.)

In certain embodiments, provided methods further comprise a step ofpurifying one or more cleavage products (i.e., free fatty acid, freeamino acids, and/or free alcohols) from residual components.

Uses

Cleavage products generated by methods of the present disclosure mayhave commercial value. For example, purified amino acids can be sold foruse in feed or food applications. For example, free fatty acids can beincorporated into personal care products, cosmetics, food, or feed.Alternatively or additionally, free fatty acids can be sold for use indiesel fuel. For example, after hydrotreatment to reduce the oxygencontent of free fatty acids, a free fatty acid such as myristic acidwill be converted to tetradecane and tridecane. These hydrocarbons havea molecular weight ideal for use as diesel fuel. Alkanes in diesel fueltypically range in size from ten to twenty-five carbons, with maincomponents having a size between 13 to 17 carbons. (See, e.g., Liang etal., “The organic composition of diesel particulate matter, diesel fueland engine oil of a non-road diesel generator,” J Environ Monit7:983-988, 2005, the entire contents of which are incorporated byreference herein.)

Alternatively or additionally, hydrocarbons obtained from cleavageproducts generated by methods of the present disclosure can be used asgasoline. Particular peptide synthetase systems that link fatty acids toamino acids have particular preferences in terms of the length of thefatty acid used in the reaction. Certain peptide synthetase enzymessynthesize acyl peptides in which the fatty acid is methylated at one ormore positions. Methylated hydrocarbons composed of 4 to 12 carbons arepreferred hydrocarbons for use as gasoline (e.g.,2,2,4-trimethylpentane), and are known to have a higher “octane rating”than straight-chain hydrocarbons of similar molecular weight. (See,e.g., Balaban et al., structure-property analysis of octane numbers forhydrocarbons (alkanes, cycloalkanes, alkenes),” MATCH Commun Math ComputChem 28:13-27, 1992.) The octane rating is as measure of the capacity ofgasoline fuel to burn effectively when intentionally ignited (forexample, via ignition due to spark from a spark plug in an automobile)and not to explode prematurely when compressed (in the absence of anintentionally created spark). Explosion in the absence of a spark causesengine “knocking”, is associated with low octane fuels, and should beavoided. (See, e.g., Perdih et al., “Chemical interpretation of octanenumber,” Acta Chim Slov 53:306-315, 2006.) The Blending Octane Number(BON) is a measure of the octane number of a single component whenblended with other components. (See, e.g., Leffler, “Petroleum refiningin nontechnical language, fourth edition,” Copyright, Penn WellCorporation, Tulsa, Okla., 2008.). Use of a peptide synthetase enzyme tolink a terminally branched fatty acid such as 4-methyl pentanoic acid toan amino acid or amino alcohol will generate a fermentation product thatcan be cleaved, by methods as described above, to produce free 4-methylpentanoic acid. Hydrotreatment of 4-methyl pentanoic acid will generatea mix of 2-methyl pentane (isohexane) and 2-methyl butane (isopentane).Isohexane and isopentane have Blending Octane Numbers (BON) of 99 and83, respectively. Hydrotreatment would be expected to generateapproximately a 50-50 mix of these species, and a 50-50 mix of thesespecies will have an octane rating of about 91 (the average of 99 and83), which is identical to the octane rating of premium gasoline (91octane). (See, e.g., Ibsen et al., “Review of market for octaneenhancers,” Final Report for NREL prepared under subcontract No.TXE-0-29113-01, 1-54, 2000.).

Thus, in some embodiments, certain methods of the present disclosurehere can be used to produce hydrocarbons that can be using as gasoline.

Acyl Alcohols

Acyl alcohols generally comprise a fatty acid moiety covalently anddirectly linked to an alcohol moiety. In some embodiments, acyl alcoholsare acyl amino alcohols, e.g., a fatty acid moiety covalently anddirectly linked to an amino alcohol moiety.

Any of a variety of acyl alcohols may be generated by compositions andmethods of the present disclosure or provided in certain methods of thepresent disclosure. By employing specific peptide synthetase domains,fatty acid linkage domains, and reductase domains in one or moreengineered polypeptides, one skilled in the art will be able to generatea specific acyl alcohol following the teachings of the presentdisclosure.

In certain embodiments, acyl alcohols generated by compositions andmethods of the present disclosure are released, by treating with areductase polypeptide, an acyl amino acid comprising an amino acidselected from one of the twenty amino acids commonly employed inribosomal peptide synthesis, i.e., alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and/or valine. In certain embodiments,acyl alcohols are released, by treating (with one or more reductasepolypeptides) an acyl amino acid comprising an amino acid other than theaforementioned twenty, for example, amino acids used less commonlyduring ribosomal polypeptide synthesis such as, without limitation,selenocysteine and/or pyrrolysine. In certain embodiments, acyl alcoholsare released from acyl amino acids that comprise amino acids that arenot used during ribosomal polypeptide synthesis such as, withoutlimitation, norleucine, beta-alanine and/or ornithine, and/or D-aminoacids.

In certain embodiments, acyl alcohols generated by certain compositionsand methods of the present disclosure, or provided in certain methods ofthe present disclosure, comprise a fatty acid moiety. A fatty acid ofacyl alcohols of the present disclosure may be any of a variety of fattyacids known to those of ordinary skill in the art. For example, acylalcohols of the present disclosure may comprise saturated fatty acidssuch as, without limitation, butyric acid, caproic acid, caprylic acid,capric acid, lauric acid, myristic acid, palmitic acid, stearicarachidic acid, behenic acid, and/or lignoceric acid. In certainembodiments, acyl alcohols of the present disclosure may compriseunsaturated fatty acids such as, without limitation, myristoleic acid,palmitoleic acid, oliec acid, linoleic acid, alpha-linolenic acid,arachidonic acid, eicosapentaenoic acid, erucic acid, and/ordocosahexaenoic acid. Other saturated and unsaturated fatty acids thatmay be used in accordance with the present disclosure will be known tothose of ordinary skill in the art. In certain embodiments, acylalcohols produced by compositions and methods of the present disclosurecomprise beta-hydroxy fatty acids as the fatty acid moiety. As isunderstood by those of ordinary skill in the art, beta-hydroxy fattyacids comprise a hydroxy group attached to the third carbon of the fattyacid chain, the first carbon being the carbon of the carboxylate group.

In some embodiments, the acyl amino alcohol is a reduction product of anacyl amino acid that comprises a naturally occurring amino acid (i.e.,alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) moiety.

In some embodiments, the acyl amino alcohol is acyl ethanolamine(“FA-ethanolamine), e.g., beta-hydroxyl myristoyl monoethanolamine(MEA), the structure of which is shown in FIG. 1. As can be seen in FIG.1, beta-hydroxyl myristoyl MEA is similar in structure to cocamide MEA,a commercially widely used nonionic surfactant.

In some embodiments, the acyl amino alcohol is acyl serinol. In someembodiments, the acyl amino alcohol is acyl alaninol.

In some embodiments, the acyl amino alcohol is a reduction product of anacyl amino acid that comprises a non-naturally occurring amino acidmoiety.

Peptide Synthetase Complexes

Peptide synthetase complexes are multienzymatic complexes found in bothprokaryotes and eukaryotes comprising one or more enzymatic subunitsthat catalyze the non-ribosomal production of a variety of peptides(see, for example, Kleinkauf et al., Annu. Rev. Microbiol. 41:259-289,1987; see also U.S. Pat. Nos. 5,652,116 and 5,795,738). Non-ribosomalsynthesis is also known as thiotemplate synthesis (see e.g., Kleinkaufet al.). Peptide synthetase complexes typically include one or morepeptide synthetase domains that recognize specific amino acids and areresponsible for catalyzing addition of the amino acid to the polypeptidechain.

The catalytic steps in the addition of amino acids include: recognitionof an amino acid by the peptide synthetase domain, activation of theamino acid (formation of an amino-acyladenylate), binding of theactivated amino acid to the enzyme via a thioester bond between thecarboxylic group of the amino acid and an SH group of an enzymaticco-factor, which cofactor is itself bound to the enzyme inside eachpeptide synthetase domain, and formation of the peptide bonds among theamino acids. A peptide synthetase domain comprises subdomains that carryout specific roles in these steps to form the peptide product. Onesubdomain, the adenylation (A) domain, is responsible for selectivelyrecognizing and activating the amino acid that is to be incorporated bya particular unit of the peptide synthetase. The activated amino acid isjoined to the peptide synthetase through the enzymatic action of anothersubdomain, the thiolation (T) domain, that is generally located adjacentto the A domain. Amino acids joined to successive units of the peptidesynthetase are subsequently linked together by the formation of amidebonds catalyzed by another subdomain, the condensation (C) domain.

Peptide synthetase domains that catalyze the addition of D-amino acidsalso have the ability to catalyze the racemization of L-amino acids toD-amino acids. Peptide synthetase complexes also typically include aconserved thioesterase domain that terminates the growing amino acidchain and releases the product, or a reductase domain that terminatesthe growing amino acid chain, reduces the terminal amino acid, andreleases the product. However, in accordance with certain embodiments ofthe present disclosure, peptide synthetase domains only contain certainof their typical subdomains, e.g., in some embodiments, peptidesynthetase domains used in accordance with the present disclosure lackthioesterase domains.

The genes that encode peptide synthetase complexes have a modularstructure that parallels the functional domain structure of thecomplexes (see, for example, Cosmina et al., Mol. Microbiol. 8:821,1993; Kratzxchmar et al., J. Bacteriol. 171:5422, 1989; Weckermann etal., Nuc. Acids res. 16:11841, 1988; Smith et al., EMBO J. 9:741, 1990;Smith et al., EMBO J. 9:2743, 1990; MacCabe et al., J. Biol. Chem.266:12646, 1991; Coque et al., Mol. Microbiol. 5:1125, 1991; Diez etal., J. Biol. Chem. 265:16358, 1990).

Hundreds of peptides are known to be produced by peptide synthetasecomplexes. Such nonribosomally-produced peptides often have non-linearstructures, including cyclic structures exemplified by the peptidessurfactin, cyclosporin, tyrocidin, and mycobacillin, or branched cyclicstructures exemplified by the peptides polymyxin and bacitracin.Moreover, such nonribosomally-produced peptides may contain amino acidsnot usually present in ribosomally-produced polypeptides such as forexample norleucine, beta-alanine and/or ornithine, as well as D-aminoacids. Additionally or alternatively, such nonribosomally-producedpeptides may comprise one or more non-peptide moieties that arecovalently linked to the peptide. As one non-limiting example, surfactinis a cyclic lipopeptide that comprises a beta-hydroxy fatty acidcovalently linked to the first glutamate of the lipopeptide. Othernon-peptide moieties that are covalently linked to peptides produced bypeptide synthetase complexes are known to those skilled in the art,including for example sugars, chlorine or other halogen groups, N-methyland N-formyl groups, glycosyl groups, acetyl groups, etc.

Typically, each amino acid of the non ribosomally-produced peptide isspecified by a distinct peptide synthetase domain. For example, thesurfactin synthetase complex which catalyzes the polymerization of thelipopeptide surfactin consists of three enzymatic subunits. The firsttwo subunits each comprise three peptide synthetase domains, whereas thethird has only one. These seven peptide synthetase domains areresponsible for the recognition, activation, binding and polymerizationof L-Glu, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Leu, the amino acidspresent in surfactin. (See FIG. 2 for schematic of the surfactinsynthetase complex.)

A similar organization in discrete, repeated peptide synthetase domainsoccurs in various peptide synthetase genes in a variety of species,including bacteria and fungi, for example srfA (Cosmina et al., Mol.Microbiol. 8, 821-831, 1993), grsA and grsB (Kratzxchmar et al., J.Bacterial. 171, 5422-5429, 1989) tycA and tycB (Weckermann et al., Nucl.Acid. Res. 16, 11841-11843, 1988) and ACV from various fungal species(Smith et al., EMBO J. 9, 741-747, 1990; Smith et al., EMBO J. 9,2743-2750, 1990; MacCabe et al., J. Biol. Chem. 266, 12646-12654, 1991;Coque et al., Mol. Microbiol. 5, 1125-1133, 1991; Diez et al., J. Biol.Chem. 265, 16358-16365, 1990). The peptide synthetase domains of evendistant species contain sequence regions with high homology, some ofwhich are conserved and specific for all the peptide synthetases.Additionally, certain sequence regions within peptide synthetase domainsare even more highly conserved among peptide synthetase domains whichrecognize the same amino acid (Cosmina et al., Mol. Microbiol. 8,821-831, 1992).

Engineered Polypeptides Useful in the Generation of Acyl Alcohols

In one aspect, provided are compositions and methods for the generationof acyl alcohols. In certain embodiments, compositions of the presentdisclosure comprise engineered polypeptides that are useful in theproduction of acyl alcohols. In certain embodiments, engineeredpolypeptides of the present disclosure comprise a peptide synthetasedomain covalently linked to a fatty acid linkage domain. In someembodiments, engineered polypeptides further comprise one or morereductase domains covalently linked to the fatty acid linkage domain andthe peptide synthetase domain. In certain embodiments, the fatty acidlinkage domain is a beta-hydroxy fatty acid linkage domain, for example,a beta-hydroxy myristic acid linkage domain.

In certain embodiments, one or more of a peptide synthetase domain, afatty acid linkage domain (e.g., a beta-hydroxy fatty acid linkagedomain), a reductase domain present in an engineered polypeptide of thepresent disclosure is naturally occurring, though their presencetogether on an engineered polypeptide, and/or their ordering, istypically not naturally occurring. Those of ordinary skill in the artwill be aware of naturally occurring polypeptides that comprise one ormore such domains, which domains can advantageously be used inaccordance with the present disclosure. A non-limiting example of anaturally occurring polypeptide synthetase complex that comprises, forexample, multiple peptide synthetase domains, a beta-hydroxy fatty acidlinkage domain and a thioesterase domain includes surfactin synthetase.Engineered polypeptides of the present disclosure may comprise one ormore of these domains that are naturally occurring in the surfactinsynthetase complex.

As will be understood by those of ordinary skill in the art afterreading this specification, it will typically be the fatty acid linkagedomain of engineered polypeptides of the present disclosure that specifythe identity of the fatty acid of the acyl amino acid. For example, thebeta-hydroxy fatty acid linkage domain of the SRFA protein of thesurfactin synthetase complex (described further, for example, in U.S.Pat. No. 7,981,685) recognizes and specifies beta-hydroxy myristic acid,the fatty acid present in surfactin. Thus, in certain embodiments,engineered polypeptides of the present disclosure comprise thebeta-hydroxy fatty acid linkage domain of the SRFA protein of thesurfactin synthetase complex, such that the acyl alcohol produced by theengineered polypeptide comprises beta-hydroxy myristic acid. The presentdisclosure encompasses the recognition that engineered polypeptides ofthe present disclosure may comprise other beta-hydroxy fatty acidlinkage domains from other peptide synthetase complexes in order togenerate other acyl alcohols.

In certain embodiments, engineered polypeptides of the presentdisclosure comprise an engineered fatty acid linkage domain (e.g. abeta-hydroxy fatty acid linkage domain) that is similar to a naturallyoccurring fatty acid linkage domain. For example, such engineered fattyacid linkage domains may comprise one or more amino acid insertions,deletions, substitutions, or transpositions as compared to a naturallyoccurring fatty acid linkage domain. Additionally or alternatively, suchengineered fatty acid linkage domains may exhibit homology to anaturally occurring fatty acid linkage domain, as measured by, forexample, percent identity or similarity at the amino acid level, forexample, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%, or at least 98% identity at the amino acidlevel. Additionally or alternatively, such engineered fatty acid linkagedomains may comprise one or more amino acid sequences that conform to aconsensus sequence characteristic of a given naturally occurring fattyacid linkage domain. In certain embodiments, an engineered fatty acidlinkage domain that is similar to a naturally occurring fatty acidlinkage domain retains the fatty acid specificity of the naturallyoccurring fatty acid linkage domain. For example, the present disclosureencompasses the recognition that one or more amino acid changes may bemade to the beta-hydroxy fatty acid linkage domain of the SRFA proteinof the surfactin synthetase complex, such that the engineeredbeta-hydroxy fatty acid linkage domain still retains specificity forbeta-hydroxy myristic acid. As will be recognized by those of ordinaryskill in the art after reading this specification, engineeredpolypeptides containing such an engineered beta-hydroxy fatty acidlinkage domain will be useful in the generation of acyl alcoholscomprising beta-hydroxy myristic acid, such as, without limitation,beta-hydroxyl myristoyl MEA.

Engineered fatty acid linkage domains may exhibit one or moreadvantageous properties as compared to a naturally occurring fatty acidlinkage domain. For example, engineered polypeptides comprising suchengineered fatty acid linkage domains may yield an increased amount ofthe acyl alcohol, may be more stable in a given host cell, may be lesstoxic to a given host cell, etc. Those of ordinary skill in the art willunderstand various advantages of engineered fatty acid linkage domainsof the present disclosure, and will be able to recognize and optimizesuch advantages in accordance with the teachings herein.

As will be understood by those of ordinary skill in the art afterreading this specification, it will typically be the peptide synthetasedomain of engineered polypeptides of the present disclosure thatspecifies the identity of the amino acid of the acyl amino acid. As onenon-limiting example, the last peptide synthetase domain from thegramicidin peptide synthetase is specific for glycine. Thus, the lastpeptide synthetase domain from gramicidin peptide synthetase can be usedin methods known in the art (e.g., as described in U.S. Pat. No.7,981,685) to construct an engineered polypeptide useful in thegeneration of an acyl amino acid that comprises a glycine moiety, which,in accordance with certain methods of the present disclosure, can betreated with one or more reductase polypeptides so that acylethanolamine (FA-ethanolamine) is released. The present disclosureencompasses the recognition that engineered polypeptides of the presentdisclosure may comprise other peptide synthetase domains, e.g., otherdomains from gramicidin peptide synthetase, and/or other domains fromother peptide synthetases, such as the surfactin synthetase complex inorder to generate certain acyl alcohols. The various domains that arepresent in a given engineered polypeptide need not be derived from thesame species.

In certain embodiments, engineered polypeptides of the presentdisclosure comprise an engineered peptide synthetase domain that issimilar to a naturally occurring peptide synthetase domain. For example,such engineered peptide synthetase domains may comprise one or moreamino acid insertions, deletions, substitutions, or transpositions ascompared to a naturally occurring peptide synthetase domain.Additionally or alternatively, such engineered peptide synthetasedomains may exhibit homology to a naturally occurring peptide synthetasedomain, as measured by, for example, percent identity or similarity atthe amino acid level, for example, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95%, or at least 98%identity at the amino acid level. Additionally or alternatively, suchengineered peptide synthetase domains may comprise one or more aminoacid sequences that conform to a consensus sequence characteristic of agiven naturally occurring peptide synthetase domain. In certainembodiments, an engineered peptide synthetase domain that is similar toa naturally occurring peptide synthetase domain retains the amino acidspecificity of the naturally occurring peptide synthetase domain. Forexample, the present disclosure encompasses the recognition that one ormore amino acid changes may be made to the last peptide synthetasedomain of the gramicidin synthetase complex, such that the engineeredpeptide synthetase domain still retains specificity for glycine. As willbe recognized by those of ordinary skill in the art after reading thisspecification, engineered polypeptides containing such an engineeredpeptide synthetase domain will be useful in the generation of acylalcohols comprising ethanolamine such as, without limitation,beta-hydroxyl myristoyl monoethanolamine.

Such engineered peptide synthetase domains may exhibit one or moreadvantageous properties as compared to a naturally occurring peptidesynthetase domain. For example, engineered polypeptides comprising suchengineered peptide synthetase domains may yield an increased amount ofthe acyl alcohol, may be more stable in a given host cell, may be lesstoxic to a given host cell, etc. Those of ordinary skill in the art willunderstand various advantages of engineered peptide synthetase domainsof the present disclosure, and will be able to recognize and optimizesuch advantages in accordance with the teachings herein.

Those of ordinary skill in the art will be aware of a variety ofnaturally occurring polypeptides that comprise a naturally occurringpeptide synthetase domain, fatty acid linkage domain, thioesterasedomain and/or reductase domain that may advantageously be incorporatedinto an engineered polypeptide of the present disclosure. For example,any of a variety of naturally occurring peptide synthetase complexes(see section herein entitled “Peptide Synthetase Complexes”) may containone or more of these domains, which domains may be incorporated into anengineered polypeptide of the present disclosure. Other non-limitingexamples of peptide synthetase complexes include surfactin synthetase,fengycin synthetase, arthrofactin synthetase, lichenysin synthetase,syringomycin synthetase, syringopeptin synthetase, saframycinsynthetase, gramicidin synthetase, cyclosporin synthetase, tyrocidinsynthetase, mycobacillin synthetase, polymyxin synthetase and bacitracinsynthetase.

In certain embodiments, one or more such domains present in anengineered polypeptide of the present disclosure is not naturallyoccurring, but is itself an engineered domain. For example, anengineered domain present in an engineered polypeptide of the presentdisclosure may comprise one or more amino acid insertions, deletions,substitutions or transpositions as compared to a naturally occurringpeptide synthetase domain, fatty acid linkage domain (e.g. abeta-hydroxy fatty acid linkage domain), and/or reductase domain. Incertain embodiments, an engineered peptide synthetase domain, fatty acidlinkage domain (e.g. a beta-hydroxy fatty acid linkage domain), and/orreductase domain present in an engineered polypeptide of the presentdisclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,60, 65, 70, 75, 80, 85, 90, 95 or more amino acid insertions as comparedto a naturally occurring domain. In certain embodiments, an engineeredpeptide synthetase domain, fatty acid linkage domain (e.g. abeta-hydroxy fatty acid linkage domain), and/or reductase domain presentin an engineered polypeptide of the present disclosure comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or more amino acid deletions as compared to a naturally occurringdomain.

In certain embodiments, an engineered peptide synthetase domain, fattyacid linkage domain (e.g. a beta-hydroxy fatty acid linkage domain),and/or reductase domain present in an engineered polypeptide of thepresent disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more amino acid substitutionsas compared to a naturally occurring domain. In certain embodiments,such amino acid substitutions result in an engineered domain thatcomprises an amino acid whose side chain contains a structurally similarside chain as compared to the amino acid in a naturally occurringpeptide synthetase domain, fatty acid linkage domain, and/or reductasedomain. For example, amino acids with aliphatic side chains, includingglycine, alanine, valine, leucine, and isoleucine, may be substitutedfor each other; amino acids having aliphatic-hydroxyl side chains,including serine and threonine, may be substituted for each other; aminoacids having amide-containing side chains, including asparagine andglutamine, may be substituted for each other; amino acids havingaromatic side chains, including phenylalanine, tyrosine, and tryptophan,may be substituted for each other; amino acids having basic side chains,including lysine, arginine, and histidine, may be substituted for eachother; and amino acids having sulfur-containing side chains, includingcysteine and methionine, may be substituted for each other.

In certain embodiments, amino acid substitutions result in an engineereddomain that comprises an amino acid whose side chain exhibits similarchemical properties to an amino acid present in a naturally occurringpeptide synthetase domain, fatty acid linkage domain (e.g. abeta-hydroxy fatty acid linkage domain), and/or reductase domain. Forexample, in certain embodiments, amino acids that comprise hydrophobicside chains may be substituted for each other. In some embodiments,amino acids may be substituted for each other if their side chains areof similar molecular weight or bulk. For example, an amino acid in anengineered domain may be substituted for an amino acid present in thenaturally occurring domain if its side chains exhibits a minimum/maximummolecular weight or takes up a minimum/maximum amount of space.

In certain embodiments, an engineered peptide synthetase domain, fattyacid linkage domain (e.g. a beta-hydroxy fatty acid linkage domain),and/or reductase domain present in an engineered polypeptide of thepresent disclosure exhibits homology to a naturally occurring peptidesynthetase domain, fatty acid linkage domain, and/or reductase domain.In certain embodiments, an engineered domain of the present disclosurecomprises a polypeptide or portion of a polypeptide whose amino acidsequence is 50, 55, 60, 65, 70, 75, 80, 85 or 90 percent identical orsimilar over a given length of the polypeptide or portion to a naturallyoccurring domain. In certain embodiments, an engineered domain of thepresent disclosure comprises a polypeptide or portion of a polypeptidewhose amino acid sequence is 91, 92, 93, 94, 95, 96, 97, 98, or 99percent identical or similar over a given length of the polypeptide orportion to a naturally occurring domain. The length of the polypeptideor portion over which an engineered domain of the present disclosure issimilar or identical to a naturally occurring domain may be, forexample, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more aminoacids.

In certain embodiments, an engineered peptide synthetase domain, fattyacid linkage domain (e.g. a beta-hydroxy fatty acid linkage domain),and/or reductase domain present in an engineered polypeptide of thepresent disclosure comprises an amino acid sequence that conforms to aconsensus sequence of a class of engineered peptide synthetase domains,fatty acid linkage domains, and/or reductase domains. For example, areductase domain may comprise the consensus sequence:[LIVSPADNK]-x(9)-{P}-x(2)-Y-[PSTAGNCV]-[STAGNQCIVM]-[STAGC]-K-{PC}-[SAGFYR]-[LIVMSTAGD]-x-{K}-[LIVMFYW]-{D}-x-{YR}-[LIVMFYWGAPTHQ]-[GSACQRHM](SEQ ID NO: 1).

In certain embodiments, an engineered peptide synthetase domain, fattyacid linkage domain (e.g. a beta-hydroxy fatty acid linkage domain),and/or reductase domain present in an engineered polypeptide is both: 1)homologous to a naturally occurring engineered peptide synthetasedomain, fatty acid linkage domain, and/or reductase domain, and 2)comprises an amino acid sequence that conforms to a consensus sequenceof a class of engineered peptide synthetase domain, fatty acid linkagedomain, and/or reductase domains.

In certain embodiments, engineered polypeptides of the presentdisclosure comprise two or more naturally occurring polypeptide domainsthat are covalently linked (directly or indirectly) in the polypeptidein which they occur, but are linked in the engineered polypeptide in anon-natural manner. As a non-limiting example, two naturally occurringpolypeptide domains that are directly covalently linked may be separatedin the engineered polypeptide by one or more intervening amino acidresidues. Additionally or alternatively, two naturally occurringpolypeptide domains that are indirectly covalently linked may bedirectly covalently linked in the engineered polypeptide, e.g. byremoving one or more intervening amino acid residues.

In certain embodiments, two naturally occurring peptide domains that arefrom different peptide synthetases are covalently joined to generate anengineered polypeptide of the present disclosure. As a non-limitingexample, engineered polypeptides of the present disclosure may comprisea beta-hydroxy fatty acid linkage domain from the SRFA protein, and apeptide synthetase and reductase domain from the gramicidin synthetasecomplex, which peptide synthetase domain and beta-hydroxy fatty acidlinkage domain are covalently linked to each other (e.g. via peptidebonds); in some embodiments, the reductase domain is also covalentlylinked to the peptide synthetase and fatty acid linkage domains.

The present disclosure encompasses engineered polypeptides comprised ofthese and other peptide synthetase domains, fatty acid linkage domains,and reductase domains from a variety of peptide synthetase complexes. Incertain embodiments, engineered polypeptides of the present disclosurecomprise at least one naturally occurring polypeptide domain and atleast one engineered domain.

In certain embodiments, engineered polypeptides of the presentdisclosure comprise one or more additional peptide synthetase domains,fatty acid linkage domains, and/or reductase domains, and still producean acyl alcohol of interest.

Reductase Polypeptides

A reductase polypeptide for use in accordance with the presentdisclosure may be any polypeptide that 1) catalyzes reduction of anamino acid, or reduction product of an amino acid (e.g., an aminoaldehyde) attached by a peptide synthetase complex and/or 2) releases aproduct of the peptide synthetase complex with the terminal residue as areduction product of an amino acid (e.g., an amino aldehyde or analcohol such as an amino alcohol) from the peptide synthetase complex.

In some embodiments, a reductase polypeptide releases the product of theengineered polypeptide as an acyl aldehyde (e.g., an acyl aminoaldehyde) or an acyl alcohol (e.g., an acyl amino alcohol). Generally,in many embodiments, the reduction from an amino acid to any aminoalcohol is a two-step reduction.

In some embodiments, a reductase polypeptide catalyzes both steps of thetwo-step reduction, e.g., the reductase polypeptide catalyzes both areduction of an acyl amino acid to an acyl amino aldehyde and asubsequent reduction of the acyl amino aldehyde to an acyl alcohol,e.g., acyl amino alcohol, and releases an acyl amino alcohol from thepeptide synthetase from which the acyl amino acid had been produced.

In some embodiments, a first reductase polypeptide catalyzes one step ofa the two-step reduction, and a second reductase polypeptide catalyzesthe second step of the two-step reduction. For example, in someembodiments, a first reductase polypeptide catalyzes a reduction of theacyl amino acid to an acyl amino aldehyde, and a second reductasepolypeptide catalyzes a reduction of the acyl amino aldehyde to an acylalcohol, e.g., acyl amino alcohol. In some embodiments, the firstreductase polypeptide releases an acyl amino aldehyde, which issubsequently reduced by the second reductase polypeptide to an acylalcohol, e.g., acyl amino alcohol. In some embodiments, the firstreductase polypeptide does not release the acyl amino aldehyde, andinstead, the second reductase polypeptide both catalyzes a reduction ofthe acyl amino aldehyde to an acyl alcohol (e.g., acyl amino alcohol)and releases the acyl alcohol product.

In some embodiments, a reductase polypeptide has an amino acid sequencethat is identical to that of a reductase polypeptide found in nature. Insome embodiments, a reductase polypeptide has an amino acid sequencethat differs from but shows an overall level of sequence identity withand/or includes one or more characteristic sequence elements of areductase polypeptide found in nature.

In some embodiments, a reductase polypeptide is an engineeredpolypeptide, for example, in that it has an amino acid sequence that hasbeen selected and/or generated by the hand of man. For example, suchengineered reductase polypeptides may comprise one or more amino acidinsertions, deletions, substitutions, or transpositions as compared to anaturally occurring reductase polypeptide. Additionally oralternatively, such engineered reductase domains may exhibit homology toa naturally occurring reductase polypeptide, as measured by, forexample, percent identity or similarity at the amino acid level, forexample, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identity at the amino acidlevel. Additionally or alternatively, such engineered reductasepolypeptides may comprise one or more amino acid sequences that conformto a consensus sequence characteristic of a given naturally occurringreductase polypeptide.

In certain embodiments, an engineered reductase polypeptide that issimilar to a naturally occurring reductase polypeptide retains theability of the naturally occurring reductase polypeptide to act on anamino acid (or an amino acid residue within an acyl amino acid) that isincorporated into a product by a peptide synthetase, catalyze areduction reaction, and/or release a reduced product (e.g., acylaldehyde or acyl alcohol) from the peptide synthetase.

Engineered reductase polypeptides may exhibit one or more advantageousproperties as compared to a naturally occurring reductase domain. Forexample, engineered reductase polypeptides, or engineered polypeptidescomprising such engineered reductase polypeptides, may yield anincreased amount of an acyl alcohol, may be more stable in a given hostcell, may be less toxic to a given host cell, etc. Those of ordinaryskill in the art will understand various advantages of engineeredreductase polypeptides of the present disclosure, and will be able torecognize and optimize such advantages in accordance with the teachingsherein.

In some embodiments, a reductase polypeptide (whether having the same ordifferent sequence than that of a naturally occurring reductasepolypeptide) is expressed from a heterologous promoter. In someembodiments, a reductase polypeptide is linked with one or more domainsor sequences with which not it is not linked in nature, and/or it islinked with one more domains or sequences with an ordering that is notfound in nature. In certain embodiments, reductase polypeptide is partof a larger polypeptide that includes a reductase domain and one or moreother domains or sequences. The larger polypeptide may itself be anengineered polypeptide or a naturally occurring polypeptide.

In some embodiments, an engineered reductase polypeptide may comprise areductase domain that has been engineered to be covalently linked withone or more other domains or sequences.

In some embodiments, a reductase polypeptide is not covalently linked toany other polypeptide domains or sequences.

As noted above, in some embodiments, a reductase polypeptide catalyzesboth steps of the two-step reduction and releases an acyl alcohol, e.g.,an acyl amino alcohol. As a non-limiting example of such a reductasepolypeptide, the final amino acid incorporated by the peptide synthetasesystem that produces mycobacterial glycopeptidedolipid is alanine.Subsequent to alanine incorporation, a reductase domain catalyzes atwo-electron reduction that can use either NADH or NADPH as a cofactor(Chlabra et al., “Nonprocessive [2+2]e off-loading reductase domainsfrom mycobacterial nonribosomal peptide synthetases,” PNAS109(15):5681-5686, 2012, the entire contents of which are hereinincorporated by reference), thereby reducing alanine to an aldehyde,alaninal. NAD(P) then dissociates, and the reductase domain is re-loadedwith NAD(P)H and reduces alaninal to alaninol. The product, havingalaninol as the terminal residue, dissociates from the peptidesynthetase complex, followed by dissociation of NAD(P).

As noted above, in some embodiments, a first reductase polypeptidecatalyzes the first step of the two-step reduction and a secondreductase polypeptide catalyzes the second step of the two-stepreduction. As a non-limiting example, the final amino acid incorporatedby the gramicidin peptide synthetase is glycine. Subsequent to glycineincorporation, a first reductase domain catalyzes a two-electronreduction that can use either NADH or NADPH as a cofactor, therebyconverting glycine into glycinaldehyde. A separate (second) reductasepolypeptide, the product of the LgrE gene, converts glycinaldehyde intoethanolamine by an NADPH-dependent reduction reaction (Schracke et al.,“Synthesis of linear gramicidin requires the cooperation of twoindependent reductases,” Biochemistry 44:8507-8513, 2005, the contentsof which are herein incorporated in their entirety).

In some embodiments in which at least two reductase polypeptides areused, at least one is covalently linked to one or more domains within anengineered polypeptide, and at least one is not covalently linked one ormore domains within an engineered polypeptide. For example, a firstreductase polypeptide may be covalently linked one or more domainswithin an engineered polypeptide, while a second reductase polypeptidemay be not covalently linked to one or more domains within an engineeredpolypeptide.

In some embodiments in which at least two reductase polypeptides areused, at least two reductase polypeptides are covalently linked to oneor more domains within an engineered polypeptide. For example, a firstreductase polypeptide and second reductase polypeptide may be covalentlylinked to other polypeptide domains (such as a fatty acid linkage domainand one or more subdomains of a peptide synthetase module) of anengineered polypeptide, and the first and second reductase polypeptidesare covalently linked to one another either directly (with no otherpolypeptide domains intervening) or indirectly (with at least one otherpolypeptide domain intervening),

In some embodiments in which at least two reductase polypeptides areused, at least two reductase polypeptides are not covalently linked toany other polypeptide domain. For example, in some embodiments, anengineered polypeptide that is useful in producing an acyl amino acid isprovided, and a first reductase polypeptide and a second reductasepolypeptide are both separate from the engineered polypeptide.

In some embodiments in which at least two reductase polypeptides areused, at least two reductase polypeptides are covalently linked to oneanother, but are not covalently linked to any other type of polypeptidedomain, e.g., a fatty acid linkage domain or any subdomain of a peptidesynthetase module. For example, a first reductase polypeptide may becovalently linked to a second reductase polypeptide, and the first andsecond reductase polypeptides are not covalently linked to any othertype of polypeptide domain.

Table 1 provides a non-limiting list amino alcohols that are present atthe terminal ends of products known to be synthesized by peptidesynthetases. References that describe the corresponding peptidesynthetases or relevant modules are also listed in Table 1. Accordingly,Table 1 represents a non-limiting list of amino alcohols for which thereis a corresponding naturally occurring reductase domain (which wouldreduce an amino acid into the amino alcohol and./or release it) withinvarious peptide synthetase complexes or modules known in the art.However, it should be noted that while the naturally occurring versionsof such amino alcohols are present at the ends of peptide chains (i.e.linked to an amino acid), such amino alcohols are never directly linkedto fatty acids (i.e., without any intervening moieties).

TABLE 1 amino alcohols in products synthesized by peptide synthetaseenzymes Reference (the entire contents of each of the below referencesare incorporated by Amino alcohol reference herein) alaninol Tatham etal., “production of mycobacterial cell wall glycopeptidolipids requiresa member of the MbtH-like protein family,” BMC Microbiology 12: 118:1-14, 2012. valinol Wiest et al., “Identification of peptaibols fromTrichoderma virens and cloning of a peptaibol synthetase,” JBC 277(23):20862-20868, 2002. phenylalaninol Wiest et al., “Identification ofpeptaibols from Trichoderma virens and cloning of a peptaibolsynthetase,” JBC 277(23): 20862-20868, 2002. leucinol Wiest et al.,“Identification of peptaibols from Trichoderma virens and cloning of apeptaibol synthetase,” JBC 277(23): 20862-20868, 2002. serinol Anderssonet al., “Acrebol, a novel toxic peptaibol produced by an Acremoniumexuviarum indoor isolate,” J. Appl. Microbiol 21: 1-15, 2009. leucinolor isoleucinol Degenkolb et al., “The Trichoderma brevicompactum clade:a separate lineage with new species, new peptaibiotics, and mycotoxins,”Mycol Progress 7(3): 177-219, 2008. tryptophanol Lee et al., “Isolationand sequence analysis of new peptaibol, boletusin, from Boletus spp.,” JPept Sci 5(8): 374-378, 1999. ethanolamine Kessler et al., “The linearpentadecapeptide gramicidin is assembled by four multimodularnonribosomal peptide synthetases that comprise 16 modules with 56catalytic domains,” J Biol Chem 279(9): 7413-7429, 2004.

However, as previously mentioned, the acyl alcohols of the presentdisclosure are not limited to acylated versions of the alcohols listedin Table 1. For example, additional reductase polypeptides may be knownin the art that act on additional amino acids, and/or additionalreductase polypeptides may be engineered to act on additional aminoacids.

In some embodiments, a reductase polypeptide is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least98% identical at the amino acid level to the reductase domain of theterminal peptide synthetase domain (module 16) of the gramicidin peptidesynthetase, e.g., that from Bacillus brevis.

In some embodiments, a reductase polypeptide is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least98% identical at the amino acid level to the reductase domain of theterminal mycobacterial glycopeptidedolipid peptide synthetase domain,such as, by way of non-limiting example, the glycopeptidedolipid peptidesynthetase domain from Mycobacterium smegmatis.

In some embodiments, a reductase polypeptide is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least98% identical at the amino acid level to the polypeptide produced fromthe Bacillus brevis LgrE gene. (See, e.g., FIGS. 6A and 6B).

In some embodiments, such as reductase polypeptide is the “second”reductase polypeptide in which both a first and a second reductasepolypeptide is used.

In some embodiments, a reductase polypeptides is characterized by thepresence of the consensus sequence:[LIVSPADNK]-x(9)-{P}-x(2)-Y-[PSTAGNCV]-[STAGNQCIVM]-[STAGC]-K-{PC}-[SAGFYR]-[LIVMSTAGD]-x-{K}-[LIVMFYW]-{D}-x-{YR}-[LIVMFYWGAPTHQ]-[GSACQRHM](SEQ ID NO: 1), where square brackets (“[ ]”) indicate amino acids thatare typically present at that position, squiggly brackets (“{ }”)indicate amino acids that amino acids that are typically not present atthat position, and “x” denotes any amino acid or a gap. X(9) for exampledenotes any amino acids or gaps for nine consecutive positions.

Compositions

In one aspect, provided are compositions comprising an acyl alcohol(such as those made by methods of the present disclosure) comprising afatty acid covalently and directly linked to an alcohol. By “directlylinked,” it is meant that there are no intervening moieties (such asamino acids) between the fatty acid and the alcohol.

In some embodiments, the fatty acid is linked to the alcohol via anamide bond.

In some embodiments, the alcohol is an amino alcohol.

In some embodiments, compositions of the present disclosure furthercomprise one or more components of an engineered microbial cell, forexample, but not limited to, intact microbial cells, macromoleculespresent in the cell wells of such cells, and/or polypeptide endogenouslyexpressed by the engineered microbial cell. In some embodiments, the oneor more components comprise an intact microbial cell.

Host Cells and Engineered Cells

Engineered polypeptides of the present disclosure may be introduced inany of a variety of host cells for the production of acyl amino acids.As will be understood by those skilled in the art, engineeredpolypeptides will typically be introduced into a host cell in one ormore expression vectors. So long as a host cell is capable of receivingand propagating such an expression vector(s), and is capable ofexpressing the engineered polypeptide(s), such a host cell isencompassed by the present disclosure. An engineered polypeptide of thepresent disclosure may be transiently or stably introduced into a hostcell of interest. For example, an engineered polypeptide of the presentdisclosure may be stably introduced by integrating the engineeredpolypeptide into the chromosome of the host cell. Additionally oralternatively, an engineered polypeptide of the present disclosure maybe transiently introduced by introducing one or more vectors comprisingthe engineered polypeptide(s) into a host cell, which vector is notintegrated into the genome of the host cell, but is neverthelesspropagated by the host cell.

In certain embodiments, a host cell is a microbial cell. In someembodiments, the microbial cell is a bacterium. Non-limiting examples ofbacteria that are useful as host cells of the present disclosure includebacteria of the genera Escherichia, Streptococcus, Bacillus, and avariety of other genera known to those skilled in the art. In certainembodiments, an engineered polypeptide of the present disclosure isintroduced into a host cell of the species Bacillus subtilis.

Microbial host cells of the present disclosure may be wild type.Alternatively, microbial host cells of the present disclosure maycomprise one or more genetic changes as compared to wild type species.In certain embodiments, such genetic changes are beneficial to theproduction of products such as acyl amino acids and/or acyl alcohols inthe microbial host. For example, such genetic changes may result inincreased yield or purity of the acyl amino acid or acyl alcohol, and/ormay endow the microbial host cell with various advantages useful in theproduction of acyl amino acids or acyl alcohols (e.g., increasedviability, ability to utilize alternative energy sources, etc.).

In certain embodiments, the present disclosure provides an engineeredmicrobial cell (e.g., an engineered bacterial cell) comprising one ormore polypeptides of the present disclosure. In some embodiments, theengineered microbial cell also expresses one or more expression vectorsfrom which one or more polypeptides can be expressed. For example, incertain embodiments, the engineered microbial cell expresses anengineered polypeptide of the present disclosure. For example, theengineered microbial cell can comprise an engineered polypeptidecomprising a fatty acid linkage domain, a peptide synthetase domain, anda first reductase polypeptide. In some embodiments, the engineeredmicrobial cell also includes a second reductase polypeptide distinctform the first reductase polypeptide.

In some embodiments, the present disclosure provides an engineeredmicrobial cell comprising one or more engineered polypeptidescollectively comprising: a fatty acid linkage domain, a peptidesynthetase domain, and one or more reductase polypeptides, which fattyacid linkage domain and peptide synthetase domain collectively canproduce an acyl amino acid, and which one or more reductase polypeptidesare collectively capable of reducing the acyl amino acid to any acylalcohol. In some embodiments, the engineered microbial cell lacks athioesterase domain, described in further detail, for example, in U.S.Pat. No. 7,981,685. In some embodiments, the engineered microbial cellis a bacterial cell, e.g., a Bacillus cell. In some embodiments, theBacillus cell is a Bacillus subtilis cell.

Engineered bacterial cells can be grown in liquid media for a period oftime. For example, for at least or about one day, at least or about twodays, at least or about three days, or at least or about four days.Typical culture volumes include, for example, about 2 mL, about 5 mL,about 10 mL, or about 25 mL, or more than about 25 mL. In someembodiments, cells are grown for about 4 days in 10 mL cultures.

In some embodiments, products (e.g., acyl alcohols such as FA-Eth) fromthe one or more polypeptides in the engineered bacterial cell aresecreted into the liquid media. Cells can be removed from the liquidmedium, e.g., by centrifugation, thus facilitating the analysis and/orisolating of any products secreted into the liquid medium. Culture mediaor subsequently processed liquids can then be analyzed by methods knownin the art, including LC-MS.

In certain embodiments, the host cell is a plant cell. Those skilled inthe art are aware of standard techniques for introducing an engineeredpolypeptide of the present disclosure into a plant cell of interest suchas, without limitation, gold bombardment and agrobacteriumtransformation. In certain embodiments, the present disclosure providesa transgenic plant that comprises an engineered polypeptide thatproduces an acyl amino acid of interest. Any of a variety of plantsspecies may be made transgenic by introduction of an engineeredpolypeptide of the present disclosure, such that the engineeredpolypeptide is expressed in the plant and produces an acyl amino acid ofinterest. The engineered polypeptide of transgenic plants of the presentdisclosure may be expressed systemically (e.g. in each tissue at alltimes) or only in localized tissues and/or during certain periods oftime. Those skilled in the art will be aware of various promoters,enhancers, etc. that may be employed to control when and where anengineered polypeptide is expressed.

Insects, including insects that are threats to agriculture crops,produce acyl amino acids that are likely to be important or essentialfor insect physiology. For example, an enzyme related to peptidesynthetases produces the product of the Drosophila Ebony genes, whichproduct is important for proper pigmentation of the fly, but is alsoimportant for proper function of the nervous system (see e.g., Richardtet al., Ebony, a novel nonribosomal peptide synthetase for beta-alanineconjugation with biogenic amines in Drosophila, J. Biol. Chem.,278(42):41160-6, 2003). Acyl amino acids are also produced by certainLepidoptera species that are a threat to crops. Thus, compositions andmethods of the present disclosure may be used to produce transgenicplants that produce an acyl alcohol of interest that kills such insectsor otherwise disrupts their adverse effects on crops. For example, anengineered polypeptide that produces an acyl alcohol that is toxic to agiven insect species may be introduced into a plant such that insectsthat infest such a plant are killed. Additionally or alternatively, anengineered polypeptide that produces an acyl alcohol that disrupts anessential activity of the insect (e.g., feeding, mating, etc.) may beintroduced into a plant such that the commercially adverse effects ofinsect infestation are minimized or eliminated. In certain embodiments,an acyl alcohol of the present disclosure that mitigates an insect'sadverse effects on a plant is an acyl alcohol that is naturally producedby such an insect. In certain embodiments, an acyl alcohol of thepresent disclosure that mitigates an insect's adverse effects on a plantis a structural analog of an acyl alcohol that is naturally produced bysuch an insect. Compositions and methods of the present disclosure areextremely powerful in allowing the construction of engineeredpolypeptides that produce any of a variety of acyl amino acids, whichacyl amino acids can be used in controlling or eliminating harmfulinsect infestation of one or more plant species.

EXAMPLES Example 1—Engineering a Bacterial Strain to ProduceFA-Ethanolamine

The present Example describes the design of an engineered bacterialstrain for use in certain methods of the disclosure to generate an acylalcohol, in this case, FA-ethanolamine. FA-ethanolamine can be used as anonionic surfactant, and its cleavage will produce ethanolamine andfatty acids as separate products.

The final amino acid incorporated by the Bacillus brevis gramicidinpeptide synthetase is glycine. (See, e.g., Kessler et al., “The linearpentadecapeptide gramicidin is assembled by four multimodularnonribosomal peptide synthetases that comprise 16 modules with 56catalytic domains.” J Biol Chem. 2004 Feb. 27: 279(9); 7413-9, theentire contents of which are herein incorporated by reference.) Thereductase domain of the gramicidin peptide synthetase catalyzes a twoelectron reduction that can use either NADH or NADPH as a cofactor,which converts glycine into glycinaldehyde. Aldehyde can be convertedinto ethanolamine by an NADPH-dependent reduction reaction catalyzed bya second enzyme such as that encoded by the LgrE gene. (See, e.g.,Schracke et al., “Synthesis of linear gramicidin requires thecooperation of two independent reductases.” Biochemistry. 2005 Jun. 14;44(23); 8507-13, the entire contents of which are herein incorporated byreference.)

Expression Construct to Generate FA-Glycine and Reduce it toFA-Glycinaldehyde

In the present Example, a fusion construct driven by the psrf promoteris generated that includes, in the following order, the condensationdomain of the first module of the Bacillus subtilis srfAA peptidesynthetase complex and the adenylation, thiolation, and reductasedomains of module 16 (the last module) of the Bacillus brevis gramicidinsynthetase. In use in accordance to certain methods of the presentdisclosure, the condensation domain will attach a fatty acid ontoglycine (which is specified by the adenylation domain of module 16),which will then be reduced by the reductase domain of module 16 toglycinaldehyde.

The lgr gene cluster is responsible for production of gramicidin in theBacillus brevis ATCC 8185 strain. The complete nucleotide sequence ofthis gene cluster is available in GenBank under accession numberAJ566197. The LgrD gene encodes a synthetase with four modules, the lastof which is “module 16” of the Bacillus brevis gramicidin synthetasementioned above.

The sequence of module 16 was analyzed and annotated using informationfrom Manavalan et al. “Molecular modeling of the reductase domain toelucidate the reaction mechanism of reduction of peptidyl thioester intoits corresponding alcohol in non-ribosomal peptide synthetases.” BMCStructural Biology. 2010, 10:1. DOI: 10.1186/1472-6807-10-1, (the entirecontents of which are herein incorporated by reference).

To define the adenylation domain, a conserved sequence in adenylationdomains, TSGSTGNPKG (SEQ ID NO: 2), was identified near the end of themodule 16 sequence. Because the adenylation domains of peptidesynthetase modules are specific for a given amino acid, the adenylationdomain in module 16 was further defined by iteratively performingsequence comparisons with various other known peptide synthetase domainsspecific for different amino acids, and discarding regions of sequencesimilarity. Sequence comparisons were performed using the ClustalW2sequence alignment tool at the EMBL-EBI website(http://www.ebi.ac.uk/Tools/msa/clustalw2/).

After initial analyses, the amino acid sequence of the condensation,adenylation, and reductase domains of module 16 (glycine module) of theBacillus brevis gramicidin synthetase complex was identified (SEQ ID NO:3). This sequence is depicted in FIG. 4.

After further analysis, the adenylation domain of module 16 (glycinemodule) of the Bacillus brevis gramicidin synthetase complex wasidentified. This sequence is depicted in FIG. 5A (amino acid sequence,SEQ ID NO: 4) and 5B (nucleotide coding sequence, SEQ ID NO: 5).

To identify fusion points to replace the condensation domain of thegramicidin synthetase glycine module (module 16) with the condensationdomain of srfAA module 1, the amino acid sequences of module 1 of thesrfAA subunit (the “FA-Glu module) from Bacillus subtilis and of thelgrD glycine module from Bacillus brevis were compared. This comparisonidentified eight potential fusion regions for replacement of thecondensation domain.

Using molecular cloning techniques known in the art, various expressionconstructs (“Construct 1”) with different fusion points are made inwhich the psrf promoter drives expression of a fusion polypeptidecontaining, in order, the condensation domain of the first module of thesrfAA subunit of Bacillus subtilis, followed by the adenylation,thiolation, and reductase domains of Bacillus brevis

Expression Construct to Reduce FA-Glycinaldehyde and Release it asFA-Ethanolamine

An expression construct (“Construct 2”) in which an lgrE gene encodingthe polypeptide of SEQ ID NO: 6 (depicted in FIG. 6A) is driven by theprsf promoter. LgrE is a reductase that will reduce glycinaldehyde toethanolamine.

Generation of Engineered Cells Producing FA-Ethanolamine

Construct 1 and Construct 2 are both introduced into Bacillus subtiliscells and co-expressed. The cells are then grown, and production ofFA-eth from such cells is measured by methods described herein and/or inU.S. Pat. No. 7,981,685.

Example 2—Validation of Quantitative Liquid Chromatography (LC/MS)Method

The present Example demonstrates the validity of a quantitative methoddeveloped by the present inventor. The method had been used by thepresent inventor to quantify FA-Glu, FA-Gly, surfactin, surfactinanalogs, and other surfactants. This same method can also be used toquantify acyl alcohols such as FA-ethanolamine (FA-Eth). Details of theLC/MS method and representative data can be found in Tseng et al.,“Characterization of the surfactin synthetase C-terminal thioesterasedomain as a cyclic depsipeptide synthase,” Biochemistry41(45):13350-13359, 2002, the entire contents of which are incorporatedby reference herein.

In the present Example, this quantitative method was used to analyze acommercial preparation of cocoyl glycinate. It was confirmed that thecocoyl glycinate surfactant is primarily composed of glycine linked tolauric acid (a 12 carbon fatty acid (C12)). In particular, it was foundthat the surfactant is composed of glycine linked to: C12 (60%), C14(33%), and C16 (7%) (FIG. 7, panel A). It was also found that the LC/MSsignal increased linearly as increasing amounts of the surfactant weremeasured (FIG. 7, panel B), and it was possible to generate a standardcurve.

Example 3—Cleavage of an Acyl Amino Acid Using Acid

The present Example illustrates cleavage of an acyl amino acid into freefatty acids and free amino acids using a method of the presentdisclosure. In the present Example, the acyl amino acid beta-myristicglutamate (FA-Glu) was successfully cleaved using acid and heat, therebyobtaining glutamate and a beta-hydroxylated fatty acids.

FA-Glu was produced by an engineered polypeptide comprising a peptidesynthetase complex by methods described in U.S. Pat. No. 7,981,685(issued on Jul. 19, 2011), the entire contents of which are hereinincorporated by reference. One thousand ng of one sample of FA-Glu wasnot treated. Nine-hundred ng of a second sample of FA-Glu was treatedwith 6N HCl at 100° C. for 24 hours. The untreated and treated samplesof FA-Glu were analyzed using an amino acid analyzer.

In the untreated sample, no amino acids were detected (FIG. 8, panel A).A small ammonia peak was observed and was due to the use of ammoniumhydroxide during preparation of the surfactant. In addition, a standardthat was co-injected with the sample eluted at a time of approximately37 minutes.

In the HCl-treated sample, a very strong glutamate peak was detected(FIG. 8, panel B). Significantly, no other amino acids were detected,indicating that the sample was very pure (free of protein contamination)and that the engineered polypeptide that produced the FA-Glu had highspecificity, exclusively linking fatty acids to glutamate (and not toother amino acids). About 250 ng of free glutamate was detected. Thus,approximately 26.32% of the injected material was detected as glutamate.On a weight basis, glutamate represents about 40% of the mass of FA-Glu.Thus, complete hydrolysis of FA-Glu is expected to release about 380 ngof glutamate.

These data suggest that about 66% of the input FA-Glu was converted tofree fatty acid plus free glutamate by treating FA-Glu with acid andheat.

Example 4—Cleavage of an Acyl Amino Acid Using an Enzyme

The present Example illustrates cleavage of an acyl amino acid into freefatty acids and free amino acids using a method of the presentdisclosure. In the present Example, the acyl amino acid beta-myristicglutamate (FA-Glu) was successfully cleaved using an enzyme, therebyobtaining glutamate and beta-hydroxylated fatty acids.

Preparation of FA-Glu Substrates

The compound FA-Glu was produced by an engineered Bacillus subtilisstrain based on Bacillus subtilis strain OKB105 Δ(upp)Spect^(R) andengineered to encode the wild-type module 1 (with specificity forglutamic acid) and an engineered module 2 with specificity for leucine.FA-Glu was produced by this engineered strain during fermentation inM9-salts media (6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl)with a final concentration of 30 g/L glycerol and 18 g/L corn-steepliquor.

Cells were removed from the fermentation broth by centrifugation. FA-Gluwas purified from the fermentation broth using a C18-derivatized silicareverse phase column (Millipore, 3 mL Sep-Pak C18 Column). The C18reverse-phase purification of FA-Glu was performed manually undervacuum. After the addition of 10 mL fermentation broth to the column,the column was washed once with water and once with 10% methanol:water.FA-Glu was eluted in 100% methanol and then dried under vacuumcentrifugation.

Enzyme Reaction Conditions

After purification, 9.6 mg of crude FA-Glu was dissolved in 1.5 mL 10%water:methanol. Using LC/MS (liquid chromatography/mass spectrometry) todetermine product peak area and using FA-Glu-Leu as the external massstandard, FA-Glu was determined to have an approximate concentration of400 μM.

Acylase I from porcine kidney (PKA I) was purchased from Sigma-Aldrich,product number A3010. The reaction buffer and enzyme diluent was 100 mMpotassium phosphate buffer, pH 7.0, as suggested by Sigma-Aldrich (SigmaQuality Control Test Procedure: M. A. Mitz and R. J. Schlueter (1958)Biochimica Et Biophysica Acta 27, 168-172). Cobalt chloride (CoCl₂) wasadded to each reaction tube to a final concentration of 1 mM. (Theonline product description (www.sigmaaldrich.com) for porcine kidneyacylase suggested including Co²⁺ or Zn²⁺ in the reaction buffer toactivate porcine kidney acylase.)

Reaction conditions for the deacylation of FA-Glu by the porcine kidneyacylase I (PKA I) are described in Table 2. One hundred-microliters ofFA-Glu corresponds to an approximate final substrate concentration of79.8 μM (3.99 μg) per reaction sample. One hundred-microliters ofacylase I corresponds to 100 μg of dried enzyme preparation per reactionsample. The digestions described in Table 1 were incubated at 25° C. for96 hrs. Samples were placed at 4° C. prior to LC/MS analysis.

TABLE 2 Reaction conditions for digestion of FA-Glu with porcine kidneyacylase I (PKA I) Total volume Reaction Substrate Enzyme BufferActivator V_(t) Reaction 1 FA-Glu, PKA I, 100 μL 299 μL 500 mM 500 μL100 μL CoCl₂, 1 μL Reaction 2 No PKA I, 100 μL 399 μL 500 mM 500 μLSubstrate CoCl₂, 1 μL Reaction 3 FA-Glu, No Enzyme 399 μL 500 mM 500 μL100 μL CoCl₂, 1 μLAnalysis of Enzymatic Digestion Products by Liquid Chromatography-MassSpectrometry (LC/MS)

Before injection into the LC/MS analyzer, enzymatic reactions werecentrifuged at 10,000×g for 5 minutes, and precipitated CoCl₂ waspelleted. The reaction supernatant was then filtered through a MilliporeUltrafree-MC 0.45-μm column during centrifugation at 5,000×g for 5minutes.

The LC system was comprised of a Thermo-Scientific Accela-autosampler,an Accela-pump, and an Accela-PDA detector. After digestion of FA-Gluwith PKA I, the Thermo Scientific C18-HPLC column Hypersil Gold was usedto separate FA-Glu from beta-hydroxy fatty acids and glutamate. Themobile phase for the reverse phase LC separation of FA-Glu frombeta-hydroxy fatty acids and glutamate was 100% water (supplemented with1% acetic acid) for 3 minutes, 100% water to 100% acetonitrile(supplemented with 1% acetic acid) in 4 minutes, 100% acetonitrile for 2minutes, and 100% isopropanol for 4 minutes. The LC system was thenre-equilibrated to 100% water for 2 minutes before the next LC/MSinjection.

After LC separation, FA-Glu and beta-hydroxy fatty acids were detectedusing a Thermo Scientific LXQ in electrospray-negative mode. The massspectrometer was programmed to capture from a first mass of m/z=100 to alast mass of m/z=1,200. Flow from the LC was diverted away from the MSdetector the first 2.5 minutes and into the MS detector for theremaining 12.5 minutes.

Glutamate was detected using a Thermo Scientific LXQ in positive mode.The mass spectrometer was programmed to capture from a first mass ofm/z=130 to a last mass of m/z=160. Flow from the LC was diverted intothe MS detector for the first 3 minutes and away from the MS detectorfor the remaining 12 minutes.

Results

FIGS. 9, 10, 11, 12, and 13 depict the LC/MS chromatograms for thedetection of beta-hydroxy fatty acid compounds and FA-Glu for m/z ratios(1) from 200 to 900, (2) from 190 to 300, (3) from 440 to 558, (4) from320 to 430, and (5) from 682 to 848, respectively. Panel A in eachfigure depicts results from Reaction 1 (FA-Glu incubated with PKA I),Panel B in each figure depicts results from Reaction 2 (PKAI incubatedwithout substrate), and Panel C in each figure depicts results fromReaction 3 (FA-Glu incubated without enzyme).

The LC/MS results confirm the formation of beta-hydroxy fatty acids fromFA-Glu after incubation with PKA I. As shown in FIG. 9, panel A themonomer of the 14-carbon (C14) beta-hydroxy fatty acid was identifiedwith the expected m/z ratio of 257.2, and the homodimer was identifiedwith the expected m/z ratio of 514.8. The monomers for the C12-, C13-,C14-, and C15-beta-hydroxy fatty acids were identified with theirexpected m/z ratios of 229.2, 243.2, 257.2, and 273.2 respectively (FIG.9, panel A and FIG. 10, panel A). The homodimers for the C13- andC14-beta-hydroxy fatty acids, and the heterodimer for theC14-/C15-beta-hydroxy fatty acids, were identified with m/z ratios of486.9, 514.8, and 530.9 respectively (FIG. 9, panel A). The homodimersfor the C13-, C14-, and the C15-beta-hydroxy fatty acids were identifiedwith m/z ratios of 486.9, 514.8, and 546.8 respectively (FIG. 11, panelA), and the heterodimers for the C13-/C14-beta-hydroxy fatty acids andC14-/C15-beta-hydroxy fatty acids were identified with m/z ratios of499.9 and 530.9, respectively (FIG. 11, panel A).

While the monomers of the beta-hydroxy fatty acids were detected with1/20^(th) the intensity during the LC/MS analysis for the incubation ofPKA I alone (FIG. 9, panel B and FIG. 10, panel B), and with ⅕^(th) to ½the intensity for the incubation of FA-Glu alone (FIG. 10, panel C),none of the homodimers or heterodimers of the beta-hydroxy fatty acidscompounds were accurately identified from either reaction sample (FIG.11, panel B and FIG. 11, panel C). The compounds with m/z ratios of455.9, 469.9, 483.9 and 499.9 (FIG. 11, panel C) are unknown compoundspurified from the fermentation broth of the 27982-H1 culture.

This data suggests that the formation of variable carbon-chain lengthbeta-hydroxy fatty acids from FA-Glu is catalyzed by PKA I and is notdue to presence of other unknown compounds in the enzyme or substratepreparations. The presence of compound peaks with the expected m/zratios for the C12-, C13-, and C14-beta-hydroxy fatty acids inelectrospray negative mode indicate that the PKA I enzyme is capable ofcleaving all carbon-chain lengths for FA-Glu.

To determine the efficiency of the digestion of FA-Glu by PKA I,substrate depletion was compared between Reaction 1 (FA-Glu and PKA I)and Reaction 3 (FA-Glu only). While only the C12-FA-Glu compound (m/zratio=358.2, FIG. 9, panel A) was detected after incubating FA-Glu withPKA I, all monomer peaks for the C12-, C13-, C14-FA-Glu substrates weredetected after incubation of FA-Glu alone (m/z ratios=344.3, 358.3,372.3, and 386.3, respectively, FIG. 9, panel C). Homodimers for C11-,C12-, C13-, and C14-FA-Glu substrates were detected with m/z ratios of716.9, 744.9, and 772.9, respectively (FIG. 9, panel C). Monomers forC15- and C16-FA-Glu-Leu had m/z ratios of 400.3 and 414.3 respectively(FIG. 12, panel C), and the homodimer of C15-FA-Glu-Leu had an m/z ratioof 800.8 (FIG. 13, panel C). Heterodimers were also detected:C11-/C12-FA-Glu (m/z ratio=702.8), C12-/C13-FA-Glu (m/z ratio=730.9),C13-/C14-FA-Glu (m/z ratio=758.9), and C14-/C15-FA-Glu (m/z ratio=786.9)(FIG. 13, panel C).

Results of the substrate depletion analysis clearly indicate thatdigestion of FA-Glu by PKA I was nearly 100% complete after 96 hours ofincubation. In addition to the C12-FA-Glu compound (m/z ratio=358.2,FIG. 9, panel A), the C11-, C12-, and C13-FA-Glu substrates were alsodetected with the m/z ratios=344.3, 372.3, and 386.3 respectively (FIG.12, panel A). None of the expected homodimers or heterodimers of theseFA-Glu substrates were detected when FA-Glu was incubated in PKA I (FIG.13, panel A), showing that the concentration of FA-Glu was too low tocause dimer formation. As for the reaction sample that contained onlyPKA I (Reaction 2), no FA-Glu monomer, homodimer, or heterodimer peakswere detected (FIG. 9, panel B; FIG. 12, panel B; and FIG. 13, panel B).

To determine the relative amount of FA-Glu remaining after incubatingFA-Glu with PKA I, Xcalibur software was used to calculate the peak areafor each of the FA-Glu compounds. Relative to the amounts observed whenFA-Glu was incubated alone, the C11-, C12-, C13-, C14-, C15, andC16-FA-Glu substrates were 91%, 64%, 97%, 99%, 100%, and 100% digested,respectively, by PKA I.

To further confirm that FA-Glu had been enzymatically digested by PKA I,LC/MS was also used to detect glutamate. A standard preparation ofglutamate was used to develop and optimize the LC/MS conditions neededto identify free glutamate generated from the digestion of FA-Glu. Thestandard preparation of glutamate was detected in electrospray positivemode with an expected m/z ratio of 148.1.

FIG. 14 depicts the LC/MS chromatograms for the detection of glutamate.Panel A depicts results from Reaction 1 (FA-Glu incubated with PKA I),Panel B depicts results from Reaction 2 (PKAI incubated withoutsubstrate), and Panel C depicts results from Reaction 3 (FA-Gluincubated without enzyme).

Glutamate was detected after incubation of FA-Glu with PKA I with an m/zratio of 148.1 (FIG. 14, panel A). Glutamate was not detected in thesample containing only PKA I (FIG. 14, panel B) or in the samplecontaining only FA-Glu (FIG. 14, panel C). Several background peaks wereobserved. Compound peaks with m/z ratios of 145.1, 146.2, 147.2, and149.2 were present in all three reaction samples; these peaks mayrepresent compounds present in the 1× reaction buffer. Background peakswith m/z ratios of 148.2 (FIG. 14, panel B) and of 148.4 (FIG. 14, panelC) may be due to the presence of either PKA I or FA-Glu, respectively;these peaks are outside the acceptable deviation for glutamatedetection, which is +/−0.1 mass units.

In summary, these results confirm that incubation of FA-Glu with PKA Iresults in cleavage of the FA-Glu into free beta-hydroxy fatty acids andglutamate.

The invention claimed is:
 1. A composition comprising an acyl aminoalcohol and an intact engineered microbial cell or one or morecomponents thereof, wherein the engineered intact microbial cell or oneor more components thereof comprises one or more polypeptides, whichcollectively comprises: a fatty acid linkage domain comprising acondensation domain of a SrfA subunit of Bacillus subtilis's surfactinsynthetase, a single peptide synthetase domain comprising an adenylationdomain of a glycine module of Bacillus brevis's gramicidin peptidesynthetase, and one or more reductase polypeptides comprising an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 1, and wherein the one or more reductase polypeptides arecollectively capable of reducing an acyl amino acid to an acyl aminoalcohol.
 2. The composition of claim 1, comprising the intact engineeredmicrobial cell.
 3. The composition of claim 1, wherein the fatty acidlinkage domain is covalently linked to the single peptide synthetasedomain.
 4. The composition of claim 3, wherein the fatty acid linkagedomain and the single peptide synthetase domain are collectively capableof producing an acyl amino acid.
 5. The composition of claim 1, whereinthe one or more reductase polypeptides are covalently linked to thefatty acid linkage domain and the single peptide synthetase domain. 6.The composition of claim 1, wherein the one or more polypeptides presentin the engineered intact microbial cell or one or more componentsthereof lack a thioesterase domain.
 7. The composition of claim 1,wherein the fatty acid linkage domain is a beta-hydroxy fatty acidlinkage domain.
 8. The composition of claim 7, wherein the beta-hydroxyfatty acid linkage domain is a beta-hydroxy myristic acid linkagedomain.
 9. The composition of claim 1, wherein the single peptidesynthetase domain comprises the adenylation domain, and a thiolationdomain, which the adenylation domain and the thiolation domain arecovalently linked.
 10. The composition of claim 1, wherein theadenylation domain is characterized by: (i) an amino acid sequence thatis at least 90% identical to the amino acid sequence of the terminaladenylation domain of the gramicidin peptide synthetase from Bacillusbrevis, wherein the adenylation domain of the gramicidin peptidesynthetase from Bacillus brevis is set forth in SEQ ID NO:
 4. 11. Thecomposition of claim 10, wherein the one or more reductase polypeptidescomprise a first reductase polypeptide characterized by: (i) an aminoacid sequence that is at least 90% identical to the amino acid sequenceof the reductase domain of the terminal peptide synthetase domain(module 16) of the gramicidin peptide synthetase from Bacillus brevis,wherein the terminal peptide synthetase domain (module 16) of thegramicidin peptide synthetase from Bacillus brevis having condensation,adenylation, and reductase domains is set forth in SEQ ID NO:
 3. 12. Thecomposition of claim 11, wherein the one or more reductase polypeptidescomprise a second reductase polypeptide characterized by: (i) an aminoacid sequence that is at least 90% identical to the amino acid sequenceof the polypeptide produced from Bacillus brevis LgrE gene as set forthin SEQ ID NO:
 6. 13. The composition of claim 1, wherein the acyl aminoalcohol is acyl ethanolamine.
 14. The composition of claim 13, whereinthe acyl ethanolamine is f3-hydroxy myristoyl ethanolamine.
 15. Thecomposition of claim 2, further comprising a liquid medium.
 16. Thecomposition of claim 1, wherein the microbial cell is a Bacillus cell.17. The composition of claim 16, wherein the Bacillus cell is a Bacillussubtilis cell.
 18. The composition of claim 1, wherein the one or morereductase polypeptides comprises (i) a first reductase polypeptide thatcatalyzes reduction of an acyl amino acid synthesized by the engineeredintact microbial cell or one or more components thereof to an acyl aminoaldehyde; and (ii) a second reductase polypeptide that catalyzesreduction of the acyl amino aldehyde to an acyl amino alcohol.